Article

Cite This: Anal. Chem. XXXX, XXX, XXX−XXX pubs.acs.org/ac

Single Fluorescent Probe Separately and Continuously Visualize H2S

and HClO in Lysosomes with Different Fluorescence Signals
Mingguang Ren,† Zihong Li,† Beibei Deng,† Li Wang,† and Weiying Lin*,†
†
Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and Chemical Engineering, School of Materials Science
and Engineering, University of Jinan, Jinan, Shandong 250022, P.R. China
*
S Supporting Information

ABSTRACT: A complicated relationship between the active small

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molecules exists in cells. On the organelle level, active small molecules

also play an important role in the maintenance of organelle functions
and roles. To investigate the relationship of biomolecules in subcellular,
it is necessary and critical to develop molecular tools that can track two
kinds of associated biomolecules within organelles with multiple
fluorescence signals. However, this is still an unmet challenge up to
date. Herein, we present the first single-fluorescent probe (Lyso-HA-
HS) that can detect oxidative (HOCl) and reductive (H2S) substances
within an organelle (lysosomes) with multiresponse signals. The
reactions of the new probe with H2S and HOCl simultaneously result
in the blue and red channels emissions, respectively, providing different
signal responses to the oxidative and reductive substances in the cellular
lysosomes. Using a single fluorescent probe, we first achieved dual-
channel imaging of the endogenous hypochlorous acid and hydrogen sulfide, respectively, in the lysosomes in the living cells.
Moreover, the highly desirable attributes of the probe Lyso-HA-HS (such as high selectivity, good membrane-permeability, and
lysosome enrichment ability) may enable it to be used in revealing the relationship of HOCl and H2S in lysosomes.

V arious types of active small molecules (reactive nitrogen

species, reactive oxygen species, reducing agents, etc.) are
present in cells, which maintain the function of cells and play a
very important role in a great deal of pathological and
physiological processes. In addition, some active small
molecules are in complex interactions. For example, HNO
generation may associate with two gas transmitters, NO and
H2S.1 In addition, H2S and Cys,2 O2− and H2O2,3 NO and
ONOO,4 H2O2 and HOCl,5 etc. show a certain correlation
between each other. In order to reveal the inter-relationship of
active small molecules in different physiological processes,
development of reporters that are capable of displaying
different signals for respective and continuous detection
related active small molecules is highly valuable.
Recently, there have been many outstanding achievements
which could respectively or continuously detect two related
active small molecules in cells with different signal models.6−14
However, all of these probes could only detect the related
active small molecules in the whole cell instead of a specific
organelle. More recently, well-designed fluorescent probes
which could track the biomolecules of interest in the specific
subcellular organelles have been constructed.15,16 However, it
is rare to describe a single fluorescent probe which could
respectively or continuously detect two related active small
molecules within organelles with different fluorescence signals.
Lysosomes, spherical-shaped and catabolic organelles, are
the major digestive compartment within cells.17,18 The original
knowledge of lysosomes was thought to be simple waste bags,
but now they are involved in many cellular processes as
advanced organelles and are considered key regulators of cell
homeostasis.19 There was experimental evidence that lysoso-
mal dysfunction was associated with the pathogenesis of
certain diseases such as Alzheimer’s disease (AD), cancer,
neurodegenerative disorders, and cardiovascular diseases.19,20
An abnormal concentration of active small molecules is
regarded as an important factor leading to dysfunction of the
lysosome.21 Therefore, real-time detection and imaging of
lysosomal biological species will help to understand the
intracellular reaction kinetics and mechanisms. Furthermore,
it further helped to formulate diagnostic and therapeutic
strategies.
Hypochorous acid (HOCl) is known to be one of the
powerful microbial agents, which can be produced in
immunological cells from hydrogen peroxide and chloride
ions mediated by myeloperoxidase (MPO), which was also
found in lysosome.22 In immune cells, HOCl plays an
important role in the defense against pathogens and micro-
organisms23,24 Due to the important biological role of
hypochlorite, some fluorescent probes for fluorescence imaging
of intracellular HOCl have been designed and reported.25−30
At the organelle level, an abnormal HOCl concentration leads
Received: November 5, 2018
Accepted: January 17, 2019
Published: January 17, 2019

© XXXX American Chemical Society A DOI: 10.1021/acs.analchem.8b05116

Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry
to an imbalance of redox in the lysosome, further leading to
lysosome dysfunction22 In addition, a high concentration of
HOCl can induce apoptosis of cultured cells by lysosomal
rupture.31
H2S was biosynthesis produced by enzymes catalyzed such
as cystathionine β-synthase (CBS),32 cystathionine γ-lyase
(CSE), 33 and 3-mercaptopyruvate sulphurtransferase
(MST).34 At the cell organelles level, CBS is also found in
the endosomal-lysosomal system.35 Furthermore, H2S also
functions in lysosome organelles. H2S can induce cell death,
which is related to the activation of calprotease protease,
lysosomal instability, and release of lysosomal protease.36
H2S and HOCl have interplaying roles in important
physiological processes. H2S and HOCl are important
mediators in brain function and disease. HOCl may contribute
to the extensive oxidative stress and oxidative damage observed
in human neurodegenerative diseases.37,38 H2S significantly
inhibits plasmodium citrullinase inactivation and protein
oxidation induced by HOCl, comparable to reduced
glutathione.39 H2S also inhibits cytotoxicity induced by
HOCl in cells, intracellular protein oxidation and lipid
peroxidation.37,38 Hence, respective or continuous detection
of H2S and HOCl would facilitate our understanding of the
interplay and cross-talk of these two species in cells.
In order to studies of the biological function of HOCl and
H2S in lysosomes, much effort has been focused on the
development of fluorescence probes to detect HOCl40−44 and
H2S45−49 in lysosomes individually. For continuous detection
of multiplex biomolecules in lysosomes, one way to solve the
problem is to use multiple fluorescent probes in one system.
However, the situation may be very complicated due to their
spectral cross-talks and the distinct localization and metabo-
lism. Therefore, to tackle the problem enumerated above, the
development of an effective single molecule that possesses
multiple recognition domains and distinct fluorescence signals
to H2S and HOCl is in high demand and critical.
Nevertheless, to our best knowledge, no single fluorescent
molecular probes that could detect HOCl and H2S in
lysosomes with multiresponse signals have been revealed in
the literature yet. To realize this goal, the major challenges
include (1) the probe should integrate multirecognition
domains, fluorescence reporters, and a lysosomal targeting
group in a single molecule; (2) fluorescence signals generated
by responding to different analytes should have a sufficient
signal interval to prevent interference between imaging
channels; (3) two reaction sites should have good selectivity
and cannot interact with each other; and (4) the probe should
be able to respond to the two analytes separately or
continuously with a reasonable signal response for different
analytes.
Herein, we reported the first lysosomes-targeted dual-
detection fluorescent probe (Lyso-HA-HS) for sensing
HOCl and H2S with different fluorescence signals. Lyso-HA-
HS exhibited a dramatic fluorescence increase in the red
channel in the presence of HOCl with excellent selectivity and
high sensitivity. By contrast, with the addition of H2S, the
probe displayed a huge fluorescence enhancement in the blue
channel. In addition, the probe can be used for continuous
detection of H2S and HOCl by fluorescence imaging in two
channels. Significantly, Lyso-HA-HS is capable of, respectively,
detecting endogenously produced H2S and HOCl by dual-
color fluorescence imaging in lysosomes with a high local-
B DOI: 10.1021/acs.analchem.8b05116
Article
ization coefficient between the probe and the lysosomal
localization dye.
■ EXPERIMENTAL SECTION
Materials and Instruments. Reaction reagents used in the
experiment and the stimulation and inhibitory reagents used in
the biological experiments were purchased from the supplier
and applied directly without further purification. The solvent
used in the purification of the compound by column
chromatography is analytically pure solvent without further
purification. The water used in both the spectral and cell
imaging experiments are twice-distilled water. The instruments
used for absorption, emission, and cell imaging are the same as
those reported in previous literature.50
Cells Culture and Cytotoxicity Assay. The protocols of
the cells culture and cytotoxicity assay are similar to our
previous report.51
Imaging of Exogenous H2S and HOCl in HeLa Cells.
After incubation for 24 h in culture dishes, HeLa cells were
washed with PBS and then treated with probe Lyso-HA-HS (5
μM) for further incubation for 20 min at 37 °C in the
incubator. Probe-loaded HeLa cells were incubated with Na2S
(50 μM) for 1 h or NaOCl (50 μM) 20 min at 37 °C after
washing with PBS. Lastly, HeLa cells were washed with PBS
three times and prepared for imaging.
Continuous Imaging of Exogenous H2S and HOCl in
HeLa Cells. After incubation for 24 h in culture dishes, HeLa
cells were washed with PBS and then treated with probe Lyso-
HA-HS (5 μM) for further incubation for 20 min at 37 °C in
the incubator. Probe-loaded HeLa cells were incubated with
Na2S (50 μM) for 1 h and then washed with PBS three times,
after that HeLa cells were further treated with the NaClO (50
μM) for 20 min. Lastly, HeLa cells were washed with PBS
three times and prepared for imaging.
Determining the Subcellular Location of Probe. For
confirming the intracellular localization of the probe, HeLa
cells or RAW 264.7 macrophage cells were used to colocalize
the probe and Lysotracker. The cells were treated with Lyso-
HA-HS (10 μM) and Lysotracker Green (0.5 μM) for 20 min
and then treated with 100 μM Na2S or 50 μM NaOCl for
another 20 min. Then the cells were washed by PBS prior to
imaging. Test the colocalization coefficient of the blue channel
(red channel) with the green channel separately.
Imaging of Endogenous H2S in HeLa Cells. HeLa cells
were divided into three groups and incubate for 24 h in culture
dishes. The first control group of HeLa cells was incubated
with 10 μM Lyso-HA-HS for 30 min. Then the cells were
washed by PBS prior to imaging. The second group is the
probe loaded HeLa cells incubated with cysteine (100 μM/
mL) for 2 h, then washed with PBS prior to imaging. For the
third group, the probe loaded HeLa cells incubated with
cysteine (100 μM/mL) and PAG (200 μM/mL) were
cultivated for 2 h under the same conditions. HeLa cells
were rinsed with PBS three times and prepared for imaging.
Imaging of Endogenous HOCl in Macrophage Cells.
RAW 264.7 macrophage cells were divided into three groups
and incubated for 24 h in culture dishes. The first control
group of RAW 264.7 cells was incubated with 10 μM Lyso-
HA-HS for 30 min. Then the cells were washed by PBS prior
to imaging. The second group is the probe loaded RAW 264.7
cells subsequently incubated with Lipopolysaccharide (LPS, 2
μg/mL) and Phorbol Myristate Acetate (PMA, 2 μg/mL) for 2
h prior to imaging. For the third group, the probe loaded RAW
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264.7 cells subsequently incubated with Lipopolysaccharide
(LPS, 2 μg/mL)/Phorbol Myristate Acetate (PMA, 2 μg/mL)
and 4-aminobenzoic acid hydrazide (ABH, 200 μM) for 2 h
prior to imaging. The cells were rinsed with PBS and prepared
for imaging.
Synthesis of Compound Lyso-HA-HS. Compound PF-5
(50 mg,0.058 mmol, 1.0 equiv) was dissolved in anhydrous
DMF (5 mL), and then NaN3 (11.7 mg, 0.18 mmol, 3 equiv)
was added to the mixture and reacted at 100 °C overnight with
an inert atmosphere of nitrogen; the reaction solution was
poured into 20 mL of water and extracted with EtOAc (3 × 30
mL). The organic layer was washed with water three times,
brine one time, and dried over Na2SO4. After filtration, the
filtrate was removed under the reduced pressure, the crude
product obtained, and the crude product was further purified
by column chromatography over silica gel eluting with
methanol/dichloromethane (v/v 1:30). The final product
Lyso-HA-HS is obtained of 47 mg (73%) as a gray solid. 1H
NMR (400 MHz, DMSO-d6), δ (ppm): δ 9.42 (s, 1H), 8.23 (s,
1H), 7.83 (dd, J = 18.3, 7.4 Hz, 2H), 7.67−7.51 (m, 2H), 7.25
(s, 1H), 7.19 (d, J = 7.9 Hz, 1H), 7.06 (d, J = 6.4 Hz, 1H),
6.65 (d, J = 7.2 Hz, 2H), 6.56 (d, J = 8.7 Hz, 1H), 6.46 (d, J =
8.7 Hz, 1H), 6.41−6.24 (m, 2H), 3.73 (s, 2H), 3.53 (s, 2H),
3.32 (dd, J = 23.9, 17.5 Hz, 10H), 3.19 (s, 2H), 2.84 (q, J =
14.3 Hz, 2H), 2.12 (s, 4H), 1.08 (t, J = 6.5 Hz, 6H). 13C NMR
(101 MHz, DMSO-d6)167.23, 163.88, 163.35, 155.15, 153.46,
153.22, 151.82, 151.59, 148.93, 144.63, 142.72, 133.81, 131.02,
130.04, 129.84, 129.45, 129.15, 124.44, 123.49, 123.15, 116.66,
115.99, 111.76, 108.78, 108.40, 107.17, 104.27, 101.80, 97.29,
66.52, 65.40, 59.87, 52.86, 48.40, 47.77, 46.46, 44.11, 26.81,
12.86. HRMS (ESI) m/z calcd for C44H44N9O7 [M + 1]+,
810.3364; found, 810.3360.
■
RESULTS AND DISCUSSION
Design and Synthesis of the Fluorescent Probe Lyso-
HA-HS. In this work, we aimed to develop a single fluorescent
probe which could respond to H2S, HOCl, and H2S/HOCl in
lysosomes using dual-channel imaging to present different
fluorescence signal patterns (see Figure 1). In order to image
two analytes in dual channeld individually, two fluorophores
linked with the corresponding recognition sites were needed in
a single probe. We choosing 7-azido-coumarin and dibenzoyl-
hydrazine-rhodamine as the platforms for imaging H2S and
Figure 1. Rational design of the probe Lyso-HA-HS reporting
lysosomal HOCl, H2S, and HOCl/H2S with different fluorescence
readouts.
C DOI: 10.1021/acs.analchem.8b05116
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HOCl in the blue channel and red channel, respectively. 7-
Aminocoumarin was produced by 7-azidocoumarin reacted
with H2S. Diformylhydrazine has been known to efficiently
react with HOCl by oxidizing diformylhydrazine into
diformyldiimide.52 Morpholine, a directing group of lysosomes,
is attached to rhodamine via a flexible chain, which can also
improve the water solubility of the probe. Combined with the
above elements, we designed and synthesized the first probe
that could detect HOCl and H2S in lysosomes with
multiresponse signals. The synthetic route of the probe
Lyso-HA-HS is shown in Scheme S-1, and the related
intermediates (shown in Scheme S-2) and the final product
were fully characterized by standard 1H NMR and 13C NMR
spectroscopy and mass spectrometry (MS).
Spectral Properties of Fluorescent Probe Lyso-HA-
HS. With Lyso-HA-HS on hand, the spectral properties of the
probe in the absence or presence of the HOCl, H2S, and
HOCl/H2S were determined. As shown in Figure S1, Lyso-
HA-HS has an obvious absorption peak at around 313 nm and
almost no absorption above 400 nm. However, the absorption
peak around 380 nm was significantly enhanced with the
addition of Na2S (a commonly used H2S source). Compared
with the control compound C2 (Figure S2), this may be
attributed to the changing of 7-azide to a 7-amino group of the
coumarin dye. This change in functional group also caused a
significant change in the fluorescence quantum efficiency. The
fluorescence quantum efficiencies of compounds C2 and C3
were 0.021 and 0.34, respectively, which was beneficial for the
construction of fluorescent probes. Furthermore, the probe
Lyso-HA-HS treated with NaOCl has apparent absorption at
550 nm, as their rhodamine dye trigger opened by HOCl
(Figure S1). The absorption spectrum of C4 also has an
obvious absorption peak at 550 nm after treated with NaOCl,
and the fluorescence intensity also increases with the addition
of NaOCl with the fluorescence quantum efficiency increasing
from 0.017 to 0.28 (Figure S3). When excited at 380 or 550
nm, the probe shows no emission at 448 or 580 nm. However,
when Na2S was added, 448 nm fluorescence will appear when
excited at 380 nm and (Figure 2a) accompanied by an obvious
fluorescence color change from colorless to blue (Figures S4
and S5). At room temperature, it only needs 3−4 min to reach
a plateau (Figure S6). While the probe is treated with gradually
increasing concentrations of HOCl and excited at 550 nm, the
probe generates a red fluorescence centered at 580 nm and is
accompanied by a fluorescent color change from colorless to
red (Figure 2b). The fluorescence intensity of the probe
reaches its maximum within seconds (Figure S7). The
emission intensity with the concentrations of Na2S (or
NaOCl) shows a linear relationship (Figures S8 and S9).
This result indicated that Lyso-HA-HS was suitable for the
quantitative determination of H2S. The detection limit was
calculated to be 3.4 × 10−7 M and 7.3 × 10−8 M to H2S and
HOCl, respectively. These results indicate that the probe Lyso-
HA-HS selectively responded to H2S and HOCl, respectively,
with different fluorescence signal patterns. Furthermore, the
probe has a large signal resolution of 132 nm between the
fluorescence peaks of H2S and HOCl responses, which reduces
mutual interference between the two fluorescent peaks and
reduces the occurrence of cross-color during cell imaging.
Moreover, we want to further explore the fluorescent signal
response mode of the probe when successively treated with
H2S and HOCl and different orders of addition of them and if
there is any effect on the fluorescence spectra. Whereas the
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Figure 2. (a) Changes of the emission spectra of Lyso-HA-HS (10
μM) with the addition of different equivalents of Na2S (0−10 equiv)
excited at 380 nm (b) The emission spectra of Lyso-HA-HS (10 μM)
with the addition of different equivalents of NaOCl (0−5 equiv)
excited at 550 nm. (c) Emission spectra of Lyso-HA-HS (10 μM)
treated with 5 equiv NaOCl and then added with different equivalents
of Na2S (0−20 equiv). (d) Emission spectra of Lyso-HA-HS (10
μM) treated with 10 equiv of Na2S and then added with different
equivalents of NaOCl (0−15 equiv).
fluorescence of the probe reaching the plateau at 580 nm after
reaction with HOCl and then treated with H2S, the
fluorescence at 448 nm also gradually enhanced with the
increase of H2S concentration (Figure 2c). In the same way,
when excited at 380 nm, a dramatic fluorescence enhancement
around 580 nm is shown with the addition of HOCl after
treating the probe with H2S reaching a plateau (Figure 2d). At
the same time, there is not much change in the fluorescence
peak of 448 nm with the addition of HOCl. Due to a minimal
spectral overlap between the emission spectral of 7-amino
coumarin and the absorption spectral of rhodamine (Figure
S10), the energy cannot be passed from the coumarin moiety
to the rhodamine section by fluorescence resonance energy
transfer (FRET). These results confirmed that although the
FRET process does not exist in the molecule, the fluorescent
molecules can respond to H2S and HOCl with different
fluorescence signal patterns. To confirm the sensing mecha-
nism, the products of Lyso-HA-HS reacted with HOCl, H2S,
and H2S/HOCl were confirmed by high-resolution mass
spectrometry (HRMS), respectively (Figures S29−S32). The
products of the reaction of Lyso-HA-HS reacted with HOCl,
H2S, and H2S/HOCl were determined to be compounds P1−
P3.
Effect of pH and Sensing Selectivity. Due to the
presence of several types of hydrolytic enzymes, the matrix in
lysosomes maintains an acidic environment (pH about 5.0).18
To monitor H2S and HOCl in lysosomes, the probe should
remain stable and can respond to analytes under an acidic
environment. With the pH of the test solution around 5.0, the
fluorescence spectra of the probe were measured with Na2S
and NaOCl added separately and continuously (Figure S11).
The experimental results show that the spectra changes were
similar to the previously tested (Figure 2), except a weakening
of the fluorescence intensity enhancement. As shown in Figure
S12, the probe Lyso-HA-HS was stable over a wide pH range
(2.8−11.3) and provides good spectral response to H2S and
HOCl at pH 5−10, indicating that the probe Lyso-HA-HS can
endure the acidity of the lysosome and has strong spectral
D DOI: 10.1021/acs.analchem.8b05116
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response to the analyte. Compared with the previously
reported literature, we found that the probe has a different
sensitivity to acidity.47 We speculate that the difference
between the sensitivity of Lyso-HA-HS probe and reported
compound to acids was mainly due to the different types of
atoms connected with the spiral nitrogen and the different test
conditions (Figure S13 and Figure S14). To study the
selectivity of Lyso-HA-HS toward H2S and HOCl, the probe
was incubated with various cations, anions, reactive oxygen/
nitrogen species, and other relevant species. As shown in
Figure S15, upon excitation at 380 nm, the fluorescence
enhancement of Lyso-HA-HS induced by H2S is more
significant than that caused by other reducing substance
(Cys, GHS, and Hcy). However, other species have little
interference with the probe emission spectra at 448 nm,
indicating that the probe is a selective sensor for H2S within
the emission wavelength of this segment. Similar to this, other
species only induce minimum perturbation in the fluorescence
spectra of probe Lyso-HA-HS at 580 nm, while HOCl elicits a
significant fluorescence increase at 580 nm (490-fold). While
HOCl elicits a large enhancement in the probe at the 580 nm
emission and a 490-fold increase in fluorescence intensity
(Figure S16), this indicates that the probe has good selectivity
for HOCl in this emission region. From what has been
discussed above, the probe has high selectivity to H2S and
HOCl in two different fluorescent emission regions, and such
good results allow the probe to perform fluorescence imaging
in living cells in different fluorescent channels.
Fluorescence Imaging of H2S and HOCl in Live Cells.
We have investigated the spectral response of the probe Lyso-
HA-HS to H2S and HOCl, the effect of pH on response, and
selectivity in vitro. The results show that the probe can detect
H2S and HOCl separately or continuously at different emission
wavelengths with well selectivity. We continue to evaluate the
capabilities of the probe to image the intracellular H2S and
HOCl. First, the cytotoxicity of the probe was tested by the
MTT assay using HeLa cell lines, and the results show that the
probe Lyso-HA-HS exhibited low cytotoxicity at low micro-
molar concentrations after a long period (24 h) (Figure S17).
Thus, the low cytotoxicity properties of the probe may render
the probe Lyso-HA-HS suitable for imaging H2S and HOCl in
living cells. Through the spectrum test, it can be known that
there is almost no overlap in the spectrum of the fluorescence
response of the H2S and HOCl, about 130 nm between two
emission peaks, which facilitates dual-channel imaging of the
two analytes of the probe. We used a dual-channel mode in the
detection of H2S and HOCl in living cells. Based on the
spectral data, the blue channel (excitation at 405 nm and the
emission collection at 425−475 nm) was set for the detection
of H2S. Meanwhile, the red channel (excitation at 561 nm and
the emission collection at 570−620 nm) was used for the
detection of HOCl. In HeLa cells only treated with Lyso-HA-
HS, almost no fluorescence is detected in both channels.
However, the blue channel shows strong fluorescence emission
when the probe-loaded HeLa cells are further treated with
Na2S (Figure 3b), but fluorescence signal still could not be
detected in the red channel, meaning that the probe Lyso-HA-
HS can be used to image H2S in the living cells in the blue
channel. Further, we tested the ability of the probe to image
HOCl in the HeLa cell in the red channel. A strong fluorescent
signal was detected in the red channel when probe-loaded
HeLa cells were further treated with NaOCl (Figure 3f). At the
same time, the enhancement of fluorescence signal in the blue
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Figure 3. Separate and continuous images of H2S and HOCl in HeLa
cells using probe Lyso-HA-HS. (a−d) Bright field (a), blue channel
(b), red channel (c) and merged image (d) of HeLa cells only
incubated with probe Lyso-HA-HS (5 μM) for 30 min. (e−h) Bright
field (e), blue channel (f), red channel (g), and merged image (h) of
HeLa cells incubated with Lyso-HA-HS (5 μM) for 30 min and then
further incubated with Na2S (50 μM) for another 1 h. (i−l) Bright
field (i), blue channel (j), red channel (k), and merged image (l) of
HeLa cells incubated with Lyso-HA-HS (5 μM) for 30 min and then
further incubated with NaOCl (50 μM) for another 20 min. (m−p)
Bright field (m), blue channel (n), red channel (o), and merged image
(p) of HeLa cells preincubated with Lyso-HA-HS (5 μM) for 30 min,
then treated with Na2S (50 μM) for another 1 h, washed with PBS
three times, and further loaded with NaOCl (50 μM) for another 20
min. (q−t) Bright field (q), blue channel (r), red channel (s), and
merged image (t) of HeLa cells preincubated with Lyso-HA-HS (5
μM) for 30 min, then treated with NaOCl (50 μM) for another 20
min, washed with PBS three times, and further loaded with Na2S (50
μM) for another 1 h.
channel was not detected. These data indicate that the probe
Lyso-HA-HS can be used to image intracellular HOCl in living
cells. We further tested the continuous identification ability of
Lyso-HA-HS to continuously respond to H2S and HOCl in
living cells. The probe-loaded HeLa cells were incubated with
Na2S for 1 h and then washed with PBS. The living cells were
further treated with the NaClO for 20 min. Significant
fluorescence signals enhancement appear in both channels
(Figure 3n,o). Adjusting the order of addition of the two
analytes can get the same result (Figure 3r,s). Thus, the overall
results demonstrate that Lyso-HA-HS can separately and
continuously monitor intracellular H2S and HOCl in two
channel imaging without interference between each other.
Morpholine, as targeting group of lysosome, can significantly
increase the probability of probe enrichment in lysosomes.53,54
In order to verify whether the probe Lyso-HA-HS will be
located in the lysosome, the probe was coincubated in HeLa
cells with commercial lysosomal dye (LysoTracker Green),
and then treated with Na2S or NaOCl, respectively. As
illustrated in Figure 4, the fluorescence signal from the blue
channel in the presence of H2S overlaid very well with the
fluorescence of LysoTracker Green. In the presence of HOCl,
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Figure 4. Probe Lyso-HA-HS and LysoTracker Green colocalization
imaging in HeLa cells: (a) brightfield image of HeLa cells, (b) from
blue channel (imaging of H2S), (c) from the red channel, (d) from
the green channel (lysosomes staining), and (e) merge of green and
blue channels. (f) Intensity profile of linear region of part e across the
HeLa cell and (g) intensity scatter plot of blue and green channels.
there is also a good overlap between the fluorescence imaging
of the red channel and the LysoTracker Green (Figure S18).
Pearson’s colocalization coefficients (describing the correlation
of intensity distribution between the two channels) were
calculated to be 0.89 and 0.86, respectively, confirming Lyso-
HA-HS localization in lysosomes of living cells.
Imaging of Endogenous H2S and HOCl in Live Cells.
According to the above experiments, the probe Lyso-HA-HS
can separately and continuously image exogenous H2S and
HOCl in different channels. We were interested in further
checking whether the probe can image endogenous H2S and
HOCl in lysosomes with a dual-color manner. Cysteine as a
precursor can produce endogenous biosynthesis H2S under the
catalysis of cystathionine β-synthase (CBS) inside the cell.2
HeLa cells were treated with 100 μM cysteine and incubated
for 1 h and then treated with 10 μM Lyso-HA-HS exhibiting a
dramatic fluorescence signal enhancement in the blue channel
compared with the HeLa cells without cysteine stimulated. To
further verify that, the enhancement of this fluorescent signal
observed above was due to endogenous H2S induction.
Propargylglycine (PAG) acts as an inhibitor and will limit
the production of endogenous H2S.55 With a comparison of
the image data, when HeLa cells are treated with cysteine (100
μM) and PAG (200 μM) at the same time, the fluorescence
intensity from the blue channel is obviously reduced indicating
that the enhancement of the fluorescent signal was indeed
caused by endogenous production of H2S. We further
investigated whether the probe Lyso-HA-HS could fluoresce
an image of endogenous HOCl in living macrophage cells in
the red channel. According to the literature, endogenous
HOCl will be produced when the RAW264.7 cells were
stimulated by phorbol myristate acetate (PMA) and lip-
opolysaccharides (LPS) together.56 A marked fluorescent
signal enhancement in the red channel (Figure 5f) was
observed in the RAW 264.7 cells, which were treated with LPS
(2 μg/mL) and PMA (2 μg/mL). In contrast, the RAW264.7
macrophage cells which were incubated with Lyso-HA-HS had
almost no fluorescent signal detected in the red channel
(Figure 5e). In order to verify that the fluorescence
enhancement is caused by the endogenous HOCl, we
performed a control experiment to inhibit the activity of
MPO enzyme using ABH as an inhibitor, thereby reducing the
concentration of endogenous HOCl.57 The intensity of the
fluorescent signal in the red channel is suppressed when the
stimulated RAW 264.7 cells were treated with 200 μM ABH,
meaning that the change of fluorescence signal in the red
Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry
Figure 5. (A) Imaging of endogenous H2S in HeLa cells using probe
Lyso-HA-HS. (a) HeLa cells only treated with the probe (10 μM) for
30 min; (b) HeLa cells stimulated with cysteine (100 μM/mL) and
then incubated with probe (10 μM) for 1 h; (c) HeLa cells stimulated
with cysteine (100 μM/mL), PAG (200 μM/mL), and then
incubated with probe (10 μM) for 1 h. (B) Imaging of endogenous
HOCl in Raw 264.7 macrophage cells using probe Lyso-HA-HS. (e)
Raw 264.7 cells only treated with the probe (10 μM) for 30 min; (f)
cells were preincubated with probe (10 μM) for 30 min and then
stimulated with LPS (2 μg/mL)/PMA (2 μg/mL) for 1 h; (g) cells
were preincubated with probe (10 μM) and treated with LPS (2 μg/
mL)/PMA (2 μg/mL) and ABH (200 μM) for 1 h.
channel was caused by endogenous HOCl. The data above
suggested that the probe Lyso-HA-HS was capable of
fluorescence imaging of endogenous H2S and HOCl in living
cells in the blue and red channels, respectively.
Furthermore, we tried to simultaneously fluorescence image
endogenous H2S and HOCl by means of dual stimulation of
RAW 264.7 cells, as treated cells with cysteine (100 μM) and
LPS/PMA (2/2 μg/mL) together. However, we only detected
the endogenous H2S in the blue channel, while endogenous
HOCl was not detected in the red channel. We speculate that
it may be due to interference caused by two stimulation
pathways. In the future work, we will further study and try to
solve this problem.
■
CONCLUSION
In summary, we have engineered the first fluorescent probe,
Lyso-HA-HS, which can simultaneously detect HOCl and H2S
in lysosomes with multiresponse signals. In addition, the probe
displays highly favorable properties; high selectivity, good
membrane-permeability, and a high colocalization coefficient.
These critical attributes enable tracking of endogenous H2S
and HOCl at lysosomes in living cells using a single
fluorescence probe for the first time. Thus, we expect that
the probe will be a powerful molecular tool for studying the
relationship between the redox balance and the function of the
lysosomal in living cells. Moreover, the rational design strategy
may be extended to construct powerful fluorescent probes for
tracking related active small molecules in other organelles.
■
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.8b05116.
Experimental procedures and additional spectra (PDF)
F DOI: 10.1021/acs.analchem.8b05116
Article
■ AUTHOR INFORMATION
Corresponding Author
*Fax: (+) 86-531-82769031. E-mail: weiyinglin2013@163.
com.
ORCID
Mingguang Ren: 0000-0003-2673-8348
Weiying Lin: 0000-0001-8080-4102
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This work was financially supported by NSFC (Grants
21472067, 21502067, 21672083, 21877048), the Natural
Science Foundation of Shandong Province, China (Grant
ZR2014BP001), Taishan Scholar Foundation (Grant TS
201511041), and the startup fund of University of Jinan
(Grants 309-10004 and 160082101).
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G DOI: 10.1021/acs.analchem.8b05116
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