Article

Cite This: Anal. Chem. 2019, 91, 11409−11416 pubs.acs.org/ac

Mitochondria-Immobilized Near-Infrared Ratiometric Fluorescent

pH Probe To Evaluate Cellular Mitophagy
Xiaoyi Li,†,‡ Yiming Hu,† Xiaohua Li,*,† and Huimin Ma*,†,‡
†
Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of
Chemistry, Chinese Academy of Sciences, Beijing 100190, China
‡
University of Chinese Academy of Sciences, Beijing 100049, China
*
S Supporting Information
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ABSTRACT: Mitochondria, powerhouses of cells, possess a weakly

alkaline environment. Various stress stimulations may lead to
mitophagy, which further gives a rise to mitochondrial acidification
Downloaded via LINKOPING UNIV on October 22, 2019 at 19:23:48 (UTC).

and disfunction. Therefore, monitoring mitochondrial pH alterations is

of great importance to better elucidate their role in the cellular
metabolism. Toward this end, a number of mitochondrial fluorescent
pH probes have been proposed, but most of them are based on
electrostatic attraction and readily leak out from the mitochondria
during mitophagy with decreased membrane potential, thus failing to
accurately measure the pH changes. In this work, we report a
mitochondria-immobilized ratiometric fluorescent pH probe, which
allows the quantitative measurements of mitochondrial pH. The probe
was designed and prepared by introducing a reactive benzyl chloride
into a positively charged near-infrared hydroxyl-hemicyanine. The
cationic property facilitates the probe to be quickly enriched into mitochondria, the hydroxyl group is responsible for producing
a reversible ratiometric fluorescence signal, and benzyl chloride is used to react with nucleophiles for immobilizing the probe in
mitochondria. Taking these advantages of the probe, the mitochondrial pH variations during mitophagy caused by rapamycin
and hypoxia have been determined quantitatively for the first time. The observed severe acidification of mitochondria under
these stimulations, together with the rationally designed probe, may be useful for studying the detailed function of mitochondria
in some bioprocesses.

In this respect, small molecular fluorescent probes have

M itochondria are cellular powerhouses that produce
adenosine triphosphate via the respiratory chain.1 They
provide the sites for many key metabolic processes, such as
fatty acid oxidation,2 iron metabolism,3 urea cycle,4 and
calcium storage.5 Under physiological conditions, mitochon-
dria maintain a weakly alkaline matrix (pH ∼ 8).6 However,
prolonged periods of hypoxia or other stress stimulation
would result in serious mitochondrial damage7 and even
mitophagy (a specific autophagic process for mitochondrial
quantity and quality control by selectively removing damaged
mitochondria).8 During mitophagy, mitochondria are first
encircled into autophagosome vesicles, and then delivered to
lysosome for bulk degradation and recycling as nutrients,
which may lead to the changes of some mitochondrial
microenvironments, such as the decrease of pH.9 In addition,
defective mitophagy is closely related to various pathological
processes, including cancer,10 neurodegenerative diseases,11
and metabolic disorder.12 Therefore, the development of an
efficient method that can accurately monitor the change in the
mitochondrial pH during mitophagy is of great importance to
understand the key functions of mitochondria under
physiological and pathological conditions.
become a powerful detection technique due to their
noninvasiveness, high sensitivity, and superior spatiotemporal
resolution capability.13−21 There are many positively charged
fluorescent probes reported for real-time visualization of
mitochondrial pH changes.15−21 However, the mitochondria-
targeting ability of these probes mainly derived from
rhodamine 123 and hemicyanine is highly dependent on the
mitochondrial membrane potential (MMP), which can reach
up to −180 mV.1 Once MMP decreases or vanishes, the
electrostatic attraction between mitochondria and the
positively charged probes will be weakened. As a result, this
kind of probe tends to leak out from mitochondria, failing to
monitor the changes of mitochondrial pH in the biological
events; moreover, the inevitable fluctuation of MMP may also
affect the accurate detection of mitochondrial pH. To solve
these problems, several novel mitochondria-immobilized
fluorescent probes have been proposed for monitoring the
change of the mitochondrial microenvironments.22−24 For
Received: June 19, 2019
Accepted: August 2, 2019
Published: August 2, 2019

© 2019 American Chemical Society 11409 DOI: 10.1021/acs.analchem.9b02782

Anal. Chem. 2019, 91, 11409−11416
Analytical Chemistry
instance, Lee et al. reported a mitochondria-immobilized off−
on fluorescent pH probe bearing triphenylphosphonium
cation and active chlorine;25 the cationic properties first
drove the probe into mitochondria via electrostatic
interaction, and subsequently, the benzyl chloride group
reacted with the nucleophiles (e.g., the thiol groups of
peptides and proteins) within mitochondria, resulting in high
immobilization in mitochondria. By virtue of this approach, a
few other mitochondria-immobilized pH probes have been
prepared.26,27 Unfortunately, these fluorescent probes are
intensity-based, limiting their application in the quantitative
detection of mitochondrial pH. Compared with an off−on
fluorescent probe, a single ratiometric one with dual emission
offers the possibility of self-calibration, which is more effective
and advantageous in excluding interferences from probe
concentration, instrumental efficiency, and environmental
conditions.28,29 To the best of our knowledge, however, no
such single mitochondria-immobilized ratiometric fluorescent
probe is available for pH measurements yet.
Herein, we present the first mitochondria-immobilized near-
infrared ratiometric fluorescent pH probe, HXPI-Cl. As shown
in Scheme 1, HXPI-Cl consists of a hemicyanine skeleton, a
Scheme 1. (A) Structures of Probe HXPI-Cl and Model
Compound HXPI and (B) pH Sensing Mechanism of
HXPI-Cl in Mitochondria
hydroxyl group, and a benzyl chloride moiety. The cationic
hemicyanine skeleton was chosen to facilitate the initial
accumulation of HXPI-Cl in mitochondria as well as the near-
infrared emission to minimize autofluorescence and biological
damage; the hydroxyl group of HXPI-Cl was predicted to
display a reversible ratiometric response to pH,30 and
introducing benzyl chloride was expected to immobilize the
probe within mitochondria. By such design, we envisioned
that HXPI-Cl would serve as a ratiometric pH probe and
remain in mitochondria even when MMP decreases or
vanishes.
■ EXPERIMENTAL SECTION
Reagents. Unless otherwise specified, all reagents,
including metal ions, thiols, H2O2, 2-morpholinoethanesul-
fonic acid (MES), and other chemicals, were purchased from
Beijing Chemical Plant, J&K Scientific Ltd., or Sigma-Aldrich
and used as received. MitoTracker Green FM (Mito Green),
rhodamine 123, and LysoTracker Green DND-26 (DND-26)
were obtained from Thermo Fisher. Rapamycin and carbonyl
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cyanide m-chlorophenylhydrazone (CCCP) were ordered
from MedChem Express. A human cervical cancer cell line
(HeLa) and Dulbecco’s modified Eagle’s media (DMEM)
were purchased from KeyGEN BioTECH Co., Ltd., Nanjing,
China. The preparation of reactive oxygen species and their
concentration determinations were following the reported
method.31 Ultrapure water (over 18 MΩ·cm) produced by a
Milli-Q reference system (Millipore) was used throughout the
experiments.
Apparatus. NMR spectra were acquired on a Bruker
Fourier-300 or a Bruker Avance III HD 400 spectrometer in
MeOD-d4 (Cambridge Isotope Laboratories). High-resolution
electrospray ionization mass spectrometry (HR-ESI-MS) and
high-resolution matrix-assisted laser desorption/ionization
mass spectrometry (HR-MALDI-MS) were conducted on an
APEX IV FTMS instrument (Bruker, Daltonics). UV−vis
absorption spectra were collected on a UV-2600 spectropho-
tometer (Shimadzu, Japan) in 1 cm quartz cells. Fluorescence
spectra were recorded on a Hitachi F-4600 spectrophotometer
in 1 cm × 1 cm quartz cells with both excitation and emission
slit widths of 10 nm and a PMT voltage of 400 V. MTT [3-
(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bro-
mide] analyses were performed on a microplate reader
(BioTek Synergy HT, U.S.A.). Confocal fluorescence images
were recorded on an FV 1200-IX83 confocal laser scanning
microscope (Olympus, Japan), and image processing was
carried out with Olympus software (FV10-ASW).
Synthesis of Fluorescent Probe HXPI-Cl. As depicted in
Scheme S1, HXPI-Cl was synthesized in two steps. First,
compound 1 (Scheme S1) was prepared following our
previous procedure with some modifications. 3 0 5-
(Hydroxymethyl)benzene-1,3-diol (63 mg, 0.45 mmol) and
triethylamine (0.20 mL) were dissolved in dry DMF (4.0 mL)
and stirred at room temperature for 10 min under argon.
Then, IR-780 (150 mg, 0.23 mmol) in dry DMF (1.0 mL)
was added slowly. The reaction mixture was warmed up to 70
°C and stirred for 4 h. The solvent was then evaporated under
reduced pressure, and the residue was purified by silica gel
chromatography (CH2Cl2/CH3OH, 10:1), affording com-
pound 1 as a blue solid (40 mg, 40%). 1H NMR (400
MHz, CD3OD; Figure S1) δ: 8.47 (d, J = 12.0 Hz, 1H), 7.74
(s, 1H), 7.51(d, J = 8.0 Hz, 1H), 7.40 (t, J = 8.0 Hz, 1H),
7.29−7.24 (m, 2H), 6.82 (s, 1H), 6.55 (s, 1H), 6.15 (d, J =
12.0 Hz, 1H), 4.74 (s, 2H), 4.10 (t, J = 8.0 Hz, 2H), 2.75 (t, J
= 8.0 Hz, 2H), 2.66 (t, J = 4.0 Hz, 2H), 1.92−1.84 (m, 4H),
1.74 (s, 6H), 1.04 (t, J = 8.0 Hz, 3H). 13C NMR (75 MHz,
CD3OD; Figure S2) δ: 172.6, 171.2, 162.2, 157.4, 142.5,
140.8, 140.7, 140.5, 135.6, 128.4, 124.7, 122.1, 122.0, 118.7,
114.6, 112.6, 110.6, 101.2, 98.9, 60.7, 49.1, 45.1, 28.3, 27.4,
23.8, 20.6, 20.3, 10.3. HR-ESI-MS: m/z calcd for
[C29H32NO3]+, 442.2377; found, 442.2374 (Figure S3).
Second, thionyl chloride (15 μL, 0.20 mmol) and dry
pyridine (17 μL, 0.20 mmol) were mixed in dry CH2Cl2 (1.0
mL), and then cooled to 0 °C. Compound 1 (39 mg, 0.070
mmol) dissolved in the mixture of dry CH2Cl2 (0.50 mL) and
dry DMF (0.10 mL) was slowly added into the above
solution. After 60 min, the reaction was quenched with water
(10 mL) and the mixture was extracted by CH2Cl2 (5.0 mL ×
3). The organic phase was evaporated under reduced pressure,
and the residue was purified by silica gel chromatography
(CH2Cl2/CH3OH, 25:1) to obtain HXPI-Cl as a blue solid
(20 mg, 62%). 1H NMR (400 MHz, CD3OD; Figure S4) δ:
8.65 (d, J = 14.8 Hz, 1H), 7.66 (s, 1H), 7.61 (d, J = 7.6 Hz,
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1H), 7.51−7.46 (m, 2H), 7.41−7.37 (m, 1H), 6.86 (d, J = 2.0
Hz, 1H), 6.74 (d, J = 2.0 Hz, 1H) 6.42 (d, J = 14.8 Hz, 1H),
4.83 (d, J = 2.4 Hz, 2H), 4.26 (t, J = 7.2 Hz, 2H), 2.80 (t, J =
6.0 Hz, 2H), 2.70 (t, J = 6.0 Hz, 2H), 1.97−1.92 (m, 4H),
1.79 (s, 6H), 1.07 (t, J = 7.2 Hz, 3H). 13C NMR (75 MHz,
CD3OD; Figure S5) δ: 176.3, 164.5, 161.6, 155.9, 144.1,
141.9, 141.7, 136.6, 131.8, 128.7, 126.4, 125.3, 122.3, 117.1,
114.3 112.8, 112.0, 102.2, 102.1, 50.2, 45.9, 41.6, 28.7, 27.1,
23.7, 20.7, 20.4, 10.2. HR-ESI-MS: m/z calcd for
[C29H31ClNO2]+, 460.2038; found, 460.2035 (Figure S6).
General Procedure for pH Detection. Stock solutions
(1.0 mM) of HXPI-Cl and HXPI were prepared in DMSO. A
series of standard pH buffer solutions were prepared by
mixing 20 mM Na2HPO4 and NaH2PO4, or NaH2PO4 and
H3PO4 at varied volume ratios (referred to as the phosphate
buffer below), and the accurate pH values were measured by a
Mettler Toledo FE28 pH meter. For spectroscopic measure-
ment, 30 μL of the stock solution of the probe was well-mixed
with 3.0 mL of the phosphate buffer, and then the mixture
was transferred to a 1 cm quartz cell to measure absorbance
against the corresponding reagent blank or fluorescence
spectra with λex = 635 nm. The ratio signal (I678/I714) was
calculated from the fluorescence intensities at 678 and 714
nm.
Culture of Cells. HeLa cells were cultured using DMEM
supplemented with 10% (v/v) fetal bovine serum (FBS)
(GIBCO) and 1% (v/v) penicillin−streptomycin in a
humidified incubator under normoxic [95% air and 5% CO2
(i.e., 20% O2)] or hypoxic (94% N2, 1% O2, and 5% CO2)
conditions.
Cytotoxicity Assay. The cytotoxicity of HXPI-Cl to HeLa
cells was examined by standard MTT assay according to the
previous report.30
Intracellular Fluorescence Imaging. HeLa cells in the
exponential phase of growth were seeded in 15 mm glass-
bottom culture dishes for 24 h to adhere before experiments.
For imaging, the growth medium was removed and replaced
with DMEM without FBS. Then, the HeLa cells were
incubated with 5.0 μM HXPI-Cl under normoxic or hypoxic
conditions at 37 °C for different periods of time. After that,
the cells were washed with DMEM for three times and cell
images were obtained using a 100× oil immersion objective
lens. The pixel intensity at least from five cells in each
fluorescence image was measured and averaged in this study.
Colocalization Experiments. HeLa cells seeded in glass-
bottom culture dishes were simultaneously incubated with
HXPI-Cl (1.0 μM) and Mito Green (500 nM) or DND-26
(1.0 μM) for 0.5 h at 37 °C in DMEM without FBS. After
being washed with DMEM for three times, the cells were
subjected to fluorescence imaging experiments with excitations
at either 488 nm (for Mito Green and DND-26) or 635 nm
(for HXPI-Cl); the corresponding fluorescence emissions
were collected at 500−550 nm (for Mito Green and DND-
26) and 650−750 nm (for HXPI-Cl), respectively. After the
uncoupling of mitochondria, the colocalization fluorescence
imaging was also performed. In brief, cells were first pretreated
for 5 h with DMEM containing HXPI-Cl (1.0 μM) and Mito
Green (500 nM); then, the media were replaced with fresh
DMEM containing CCCP (10.0 μM) and the cells were
incubated for another 3 h; finally, the cells were washed and
colocalization imaging was performed as mentioned above.
Intracellular pH Calibration. HeLa cells were pretreated
with DMEM containing HXPI-Cl (5.0 μM) at 37 °C for 5 h.
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Then, the media were replaced with high-K+ buffer (30 mM
NaCl, 120 mM KCl, 1.0 mM CaCl2, 0.50 mM MgSO4, 1.0
mM NaH2PO4, 5.0 mM glucose, 20 mM sodium acetate, 20
mM HEPES, 10 μM nigericin, and 20 mM MES) of various
pH values (pH 5.0−8.0). After 15 min of incubation with the
cells, the images of the green (655−685 nm) or red (695−725
nm) channel were recorded with excitation at 635 nm through
a 100× 1.4 NA objective. An intracellular pH calibration curve
(Igreen/Ired vs pH) was then constructed.
■ RESULTS AND DISCUSSION
Spectroscopic Response of HXPI-Cl to pH. The
fluorescence behavior of HXPI-Cl in various pH phosphate
buffer solutions (20 mM) was studied. As shown in Figure 1A,
Figure 1. (A) Absorption and (B) fluorescence emission spectra of
HXPI-Cl (10 μM) in the phosphate buffer (20 mM) with various pH
values. (C) Plot of I678/I714 vs pH in the range of 2.5−12. Inset: the
linear fitting curve of I678/I714 vs pH from 4.6 to 6.6. (D) pH
reversibility of HXPI-Cl between pH 4.0 and 8.0. λex = 635 nm. Data
are expressed as the mean ± SD of three separate measurements.
the absorption maximum of HXPI-Cl shifts from 610 to 694
nm as the pH value increases from 4.5 to 8.5 with an obvious
color change of the solutions from blue to green. Meanwhile,
the fluorescence intensity at 678 nm (I678) undergoes slight
decrease but that at 714 nm (I714) displays dramatic increase
(Figure 1B). This provides the basis for ratiometric measure-
ments of pH with I678/I714 (Figure 1C). Moreover, there is an
excellent linear relationship between I678/I714 and pH over the
range of 4.6−6.6 (Figure 1C, I678/I714 = 6.97−1.01 pH, r2 =
0.988). Additionally, the probe, with a pKa value of 5.77,
shows satisfactory reversibility between pH 4.0 and pH 8.0
(Figure 1D). These unique properties enable HXPI-Cl to be
feasible to accurately measure the pH changes in living cells.
Next, the selectivity of the probe was examined over various
other coexisting species. As shown in Figure S7, I678/I714 is
independent of the tested common metal ions (K+, Ca2+,
Mg2+, Cu2+, Zn2+), anions (CN−, OCl−), and redox substances
(glutathione, cysteine, H2O2, ·OH, and ONOO−) in the
phosphate buffer (20 mM, pH 7.4, 37 °C). These results
indicate that HXPI-Cl may be applied to measuring
intracellular pH with high selectivity.
Mitochondria-Targeting Ability of HXPI-Cl. Before
intracellular experiments, a standard MTT assay was carried
out to evaluate the biocompatibility of HXPI-Cl. It was found
that HXPI-Cl displayed negligible cell toxicity since nearly
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Analytical Chemistry
90% of HeLa cells survived even when the concentration of
HXPI-Cl reached 10 μM (Figure S8).
To study the mitochondria-targeting ability of the probe,
HeLa cells were costained with HXPI-Cl and Mito Green (a
commercial mitochondria-targeting dye). As shown in Figure
2, the filament-shaped mitochondria could be clearly
Figure 2. Colocalization experiment of HXPI-Cl and Mito Green in
HeLa cells. Images of HeLa cells stained with (A) Mito Green (λex =
488 nm, λem = 500−550 nm) and (B) HXPI-Cl (λex = 635 nm, λem =
650−750 nm). (C) Merged image of panels A and B. (D) Enlarged
image of the square region in panel A. (E) Enlarged image of the
square region in panel B. (F) Corresponding differential interference
contrast (DIC) image. (G) Intensity correlation plot of Mito Green
and HXPI-Cl (Pearson’s coefficient, 0.86). (H) Intensity profiles of
Mito Green and HXPI-Cl within the linear ROI (region of interest)
in image C. Scale bars = 10 μm.
visualized after staining by HXPI-Cl (Figure 2B), which is
comparable with those stained by Mito Green (Figure 2A).
Moreover, the fluorescence distribution of HXPI-Cl overlaps
well with that of Mito Green (Pearson’s coefficient, 0.86;
Figure 2, parts G and H). On the contrary, the overlap
between HXPI-Cl and DND-26 (a commercial lysosome-
targeting dye) is rather poor (Pearson’s coefficient, 0.71;
Figure S9). This explicit difference confirms the superior
mitochondria-targeting ability of HXPI-Cl.
Mitochondria-Immobilized Ability of HXPI-Cl. As
mentioned above, traditional electrostatic attraction based
probes readily leak out from mitochondria once MMP
decreases or vanishes, which may hamper their application
in monitoring the function of mitochondria in the cell-
depolarization associated events.32 To assess the immobilizing
ability of HXPI-Cl in mitochondria, a mitochondrial
uncoupling experiment was conducted. Specifically, HeLa
cells were incubated with HXPI-Cl in DMEM for 5 h. Then,
the medium was removed and replaced with DMEM
containing CCCP (a reagent that can uncouple MMP by
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rapid acidification of mitochondria and induce mitopha-
gy).33,34 Moreover, HXPI, a model compound that has no
benzyl chloride moiety but possesses the same skeleton as
HXPI-Cl (Scheme 1B), was chosen for comparison. As can be
seen from Figure 3A, the fluorescence intensity of the HXPI-
Figure 3. Mitochondria-immobilized properties of HXPI-Cl. (A) The
effect of CCCP (10 μM) on the fluorescence imaging of 5.0 μM
HXPI-Cl or HXPI in HeLa cells. λex = 635 nm, λem = 650−750 nm.
Scale bar = 10 μm. (B) HR-MALDI-MS of the reaction solution of
HXPI-Cl (10 μM) and glutathione (1.0 mM, about physiological
concentration).
Cl-stained cells remains unchanged even after a 3 h incubation
with CCCP (image c) that can cause the vanishment of MMP
(Figure S10); in contrast, a large decrease in the fluorescence
intensity is observed for the HXPI-treated cells (image i in
Figure 3A). Furthermore, similar fluorescence changes were
found in the fixed mitochondria-uncoupled HeLa cells
(treated with 3.7% formaldehyde at 4 °C for 30 min; images
e and k in Figure 3A). The above results indicate that HXPI-
Cl can be well-immobilized in mitochondria, independent of
the MMP changes. More notably, the colocalization imaging
of HeLa cells with HXPI-Cl and Mito Green or DND-26 after
depolarization (Figure S11) showed that HXPI-Cl was highly
overlapped not only with Mito Green (Pearson’s coefficient,
0.88) but also with DND-26 (Pearson’s coefficient, 0.89). The
high overlap coefficient35 between HXPI-Cl and DND-26
implies the occurrence of mitophagy, which further shows the
usefulness of HXPI-Cl in monitoring the state of mitochon-
dria in cell depolarization associated events.
Interestingly, compared with the filamentous shapes of
mitochondria depicted in Figure 2B, more bright spots
appeared when the cells were incubated with CCCP (image
c in Figure 3A). It has been reported that the outer membrane
of mitochondria may participate in autophagosome bio-
genesis.36 Therefore, we speculated that these ovoid structures
might be the destroyed mitochondria due to the severe
CCCP-induced mitophagy. It should be noted that a small
number of fluorescent dots were still observed in HeLa cells
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without CCCP (images a and g in Figure 3). Considering that
the whole incubation of cells was carried out in DMEM
without FBS for more than 8 h, mild starvation-induced
autophagy37 might also occur. This phenomenon is consistent
with the previous reports in literature.25,38 The detailed
changes in mitochondrial morphology revealed by HXPI-Cl
further prove that the probe can be immobilized in
mitochondria regardless of the MMP fluctuation.
Moreover, the immobilizing ability of HXPI-Cl and Mito
Green in mitochondria was compared under the same
conditions since both of them possess a benzyl chloride
functional group, and HXPI without the benzyl chloride
moiety was used as a reference. As shown in Figure S12, the
fluorescence intensity of Mito Green (green channel)
becomes much weaker in the presence of HXPI-Cl, which
may be ascribed to the competitive reaction of the two
compounds with the nucleophiles in mitochondria; in
contrast, HXPI fails to cause such a large decrease in the
fluorescence intensity (compare images D and H in Figure
S12). Besides, from the red channel (namely, the HXPI-Cl or
HXPI channel), the fluorescence intensity of HXPI-Cl is much
stronger than that of HXPI (compare images B and F in
Figure S12). All the above results indicate that HXPI-Cl
exhibits a much stronger mitochondria-immobilized ability
than HXPI without the benzyl chloride moiety, demonstrating
that the active chlorine plays a key role in the reaction with
intracellular nucleophiles.
To further confirm the chemical reactivity of HXPI-Cl
toward the nucleophiles such as a thiol, an in vitro experiment
was carried out using glutathione (GSH) as a model. Briefly,
HXPI-Cl and GSH were mixed in the phosphate buffer (20
mM) and stirred at 37 °C for 1 h. The reaction solution was
then extracted with CH2Cl2 and concentrated before HR-
MALDI-MS analysis. As depicted in Figure 3B (see also
Figure S13), the mass peak at m/z = 731.3121 is identified to
be the characteristic molecular ion peak of HXPI-GSH in the
reaction solution. From the above data in Figures 2 and 3, it is
concluded that HXPI-Cl can be well-immobilized within
mitochondria via the covalent attachment.
Intracellular pH Calibration with HXPI-Cl. To
quantitatively determine the mitochondrial pH, a valid
intracellular pH calibration curve should be established.
HeLa cells were pretreated with HXPI-Cl (5.0 μM) for 5 h.
Then, the medium was replaced with the high-K+ buffer of
various pH values,30 and the fluorescence signal of HXPI-Cl
was recorded in the green (655−685 nm) and red (695−725
nm) channel, respectively, to obtain the pH calibration curve
(Igreen/Ired vs pH). As shown in Figure S14, the fluorescence
intensity from the green channel gradually decreases as the pH
value changes from 5.02 to 8.09, whereas that from the red
channel remains nearly unchanged. The merged images of the
green and red channels exhibit a distinct pH-dependent
pattern (Figure 4A; see Figure S14 for more data). The
change in the ratio of fluorescence intensity from the two
channels shows a pH-dependent linear response over the pH
range of 5.02−8.09 (Figure 4B, R = 1.09−0.083 pH, r2 =
0.985), which is different from that in the extracellular
experiment but basically covers the pH range of the normal,
damaged, or pathogenic state of mitochondria in living cells.26
Moreover, HXPI-Cl displays good reversibility in HeLa cells
between pH 4.05 and 8.09 (Figure 4C). These results suggest
that HXPI-Cl may be used to quantitatively track and visualize
the mitochondrial pH change regardless of MMP variations.
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Figure 4. Calibration of intracellular pH in HeLa cells with HXPI-Cl.
(A) Merged and ratio images of the green (655−685 nm) and red
(695−725 nm) channels. Scale bar = 10 μm. (B) Linear relationship
between the ratio value (R = Igreen/Ired) and pH in HeLa cells (see
Figure S14 for more data). (C) Reversibility of HXPI-Cl between pH
4.05 and pH 8.09 in HeLa cells. λex = 635 nm. The data are
expressed as the mean ± SD of three measurements.
Monitoring the Mitochondrial pH Change during
Mitophagy Induced by Rapamycin. Application of HXPI-
Cl to determining the pH change of mitochondria during
mitophagy was first demonstrated with a rapamycin model.
Rapamycin is a widely used macrolide antibiotic which
induces mitophagy in a variety of cell types.39,40 Although
the acidification of mitochondria has been observed during
the mitophagy, it remains unknown how the exact pH values
vary. Using probe HXPI-Cl, we studied this issue. HeLa cells
were treated with 5.0 μM HXPI-Cl for 5 h, and then subjected
to rapamycin (5.0 μM) to induce mitophagy.41 Subsequently,
fluorescence images were recorded at different time points. As
shown in Figure 5A, compared with the control group, the
merged images of the rapamycin-treated HeLa cells exhibit
more obvious color change (more yellow in color) as well as
less filaments (similar to the above observation with the
CCCP treatment; image c in Figure 3A) with the increase of
incubation time, which is ascribed to the rapamycin-induced
decrease of the mitochondrial pH and the generation of
mitophagy. By using the constructed pH calibration curve, the
pH alteration in mitochondria can be quantitatively
determined. The average pH value of mitochondria in normal
HeLa cells was found to be 8.04 ± 0.13, consistent with the
value reported in earlier studies.6,25 However, the rapamycin-
induced mitophagy caused the decrease of the mitochondrial
pH value to 7.44 ± 0.33, 6.48 ± 0.26, and 5.58 ± 0.49 with
the incubation time of 3, 15, or 24 h (Figure 5B), respectively.
Such a quantitative acidification process has not been
monitored in real time yet. In addition, to exclude the
possible influence from cell starvation, another control
experiment was performed. As can be seen from Figure
S15A, the intracellular pH in the control group is nearly
unchanged with time, suggesting a neglectable contribution to
the pH alteration from cell starvation. These results indicate
the capability of HXPI-Cl in determining the pH change of
mitochondria in living cells during mitophagy.
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Anal. Chem. 2019, 91, 11409−11416
Analytical Chemistry Article

Figure 5. Confocal fluorescence images of HeLa cells during the rapamycin-induced mitophagy. (A) Green (λem = 655−685 nm) and red (λem =
695−725 nm) channels were collected at λex = 635 nm. Scale bar = 10 μm. (B) pH change of mitochondria with time. The data are expressed as
the mean ± SD of three measurements.

Figure 6. Confocal fluorescence images of HeLa cells during the hypoxia-induced mitophagy (94% N2, 1% O2, and 5% CO2). (A) Green (λem =
655−685 nm) and red (λem = 695−725 nm) channels were collected at λex = 635 nm. Scale bar = 10 μm. (B) pH change of mitochondria with
time. The data are expressed as the mean ± SD of three measurements.

Monitoring the Mitochondrial pH Change during
Mitophagy Induced by Hypoxia. It has been reported that
hypoxia may cause mitochondrial damages, and even
mitophagy.7 However, the specific change of mitochondrial
pH during the hypoxia-induced mitophagy has not been
monitored quantitatively. Here, we made such an attempt
using probe HXPI-Cl. In this experiment, the cells were
incubated under hypoxic conditions of 1% O2. As shown in
Figure 6 and Figure S15B, large fluorescence difference can be
observed between the images of the cells incubated under
11414
normoxic and hypoxic conditions. On the basis of the pH
calibration curve (Figure 4B), it can be calculated that, after
incubating under the hypoxic condition for 3, 9, or 15 h, the
mitochondrial pH value was decreased to 7.20 ± 0.12, 6.14 ±
0.46, and 5.54 ± 0.42, respectively. Such a quantitative
changing behavior has not been reported, though the
acidification of mitochondria was observed during the
hypoxia-induced mitophagy.35 The above experiments dem-
onstrate that HXPI-Cl may be also useful for the quantitative
DOI: 10.1021/acs.analchem.9b02782
Anal. Chem. 2019, 91, 11409−11416
Analytical Chemistry
measurement of mitochondrial pH variations under hypoxic
conditions.
■
CONCLUSIONS
In summary, by incorporating benzyl chloride into a stable
hemicyanine skeleton, we have developed HXPI-Cl as a new
mitochondria-immobilized near-infrared ratiometric fluores-
cent pH probe, which permits the real-time and quantitative
monitoring of pH changes associated with the mitochondrial
acidification and fusion during mitophagy. We believe that,
owing to its synthetic convenience and ratiometric feature,
HXPI-Cl may serve as a promising tool for studying the roles
of mitochondria in various intracellular pathological processes.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.anal-
chem.9b02782.
Synthetic route of HXPI-Cl, 1H and 13C NMR spectra,
additional spectroscopic data, cytotoxicity assay, and
fluorescence imaging of HeLa cells (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail: lixh@iccas.ac.cn.
*E-mail: mahm@iccas.ac.cn.
ORCID
Xiaohua Li: 0000-0001-5656-4444
Huimin Ma: 0000-0001-6155-9076
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We are grateful for the financial support from the NSF of
China (nos. 21775152, 91732104, 21535009, 21820102007,
and 21621062).
■
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