Turn-on Fluorescent Probe for Exogenous and Endogenous Imaging of Hypochlorous Acid in Living Cells and Quantitative Application in Flow Cytometry

Article abstract of DOI:10.1021/acs.analchem.7b02613

Hypochlorous acid (HClO) acts as a dominant microbicidal mediator in the natural immune system, and the excess production of hypochlorites is related to a series of diseases. Thus, it is vitally important and necessary to develop a highly sensitive and se

Full text of DOI:10.1021/acs.analchem.7b02613

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Article
A novel turn-on fluorescent probe for exogenous and endogenous imaging of
hypochlorous acid in living cells and quantitative application in flow cytometry
Zixuan Zhan, Rui Liu, Li Chai, Qiuyan Li, Kexin Zhang, and Yi Lv
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02613 • Publication Date (Web): 01 Aug 2017
Downloaded from http://pubs.acs.org on August 1, 2017
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A novel turn-on fluorescent probe for exogenous and
endogenous imaging of hypochlorous acid in living cells and
quantitative application in flow cytometry
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Zixuan Zhan, † Rui Liu, † Li Chai, ‡ Qiuyan Li, † Kexin Zhang,† Yi Lv† *
† Key Laboratory of Green Chemistry & Technology, Ministry of Education, College of
Chemistry, Sichuan University, Chengdu, Sichuan 610064, China
‡ Core Facility of West China Hospital, Sichuan university, Chengdu, Sichuan, 610041, China
*Corresponding Author. Email: lvy@scu.edu.cn; Tel. & Fax +86-28-8541-2798
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ABSTRACT: Hypochlorous acid (HClO) acts as a dominant microbicidal mediator in the
natural immune system and the excess production of hypochlorites is related to a series of
diseases. Thus, it is vitally important and necessary to develop a highly sensitive and selective
method for HClO detection in living systems and most of fluorescent probes are mainly focused
on cells imaging. Besides, accurate HClO quantitative information about individual cells in a
large cell population is extremely important for understanding inflammation and cellular
apoptosis as well. In our work, a turnꢀon fluorescent probe has been synthesized, which can
selectively and sensitively detect HClO with fast response time. The probe is almost
nonfluorescent possibly due to both the spirolactam form of fluorescein and unbridged C=N
bonds which can undergo nonꢀradiative decay process in the excited state. Upon the addition of
ClOꢀ, the probe was oxidized to ringꢀopened fluorescent form and the fluorescence intensity was
greatly enhanced. In live cell experiments, the probe was successfully applied to image
exogenous ClOꢀ in Hela cells and endogenous HClO in RAW 264.7 macrophage cells. In
particular, the quantitative information of exogenous and endogenous HClO can also be
acquired in flow cytometry. Therefore, the probe can not only image exogenous and endogenous
HClO but also provide a new and promising platform to quantitative detect HClO in flow
cytometry.
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INTRODUCTION SECTION
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It is known that reactive oxygen species (ROS) and reactive nitrogen species (RNS) cause
oxidative and nitrosative stresses, respectively. These stresses play vital roles in a diversity of
biological processes and are also connected to many diseases, including cancer and
neurodegenerative disorders.1ꢀ4 Among various ROS, hypochlorous acid (HClO) and its
conjugate base hypochlorite (ClOꢀ) are extensively employed as bleaching agents in our daily
life and also act as defense tools in the immune system.5ꢀ7 Endogenous HClO can be produced
by the reaction of hydrogen peroxide (H2O2) and chloride ion (Clꢀ) catalyzed by the heme
enzyme myeloperoxidase (MPO), which is mainly located in leukocytes including macrophages,
monocytes, and neutrophils.8ꢀ11 However, the excess production of hypochlorites from
phagocytes can lead to inflammationꢀassociated tissue damage and a series of diseases, such as
lung injury, hepatic ischemiaꢀreperfusion injury, neuron degeneration, arthritis, cardiovascular
disease and even cancer.12ꢀ15 Therefore, the development of a highly sensitive and selective
hypochlorite detection and imaging method among H2O2 and other ROS in living systems is
vitally important and necessary to unravel the fundamental processes in immune system
responses.
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Toward this end, a number of analytical methods for the detection of ClOꢀ have been
developed, including colorimetric, electrochemical, chemiluminescence methods.16ꢀ19 In
addition, compared with other detection methods, fluorescence imaging methods are generally
superior in terms of sensitivity, simplicity, response time, spatial and temporal resolution. These
innate advantages can offer applications for not only in vitro assays but also in vivo imaging
studies. So far, a number of fluorescent probes for specific detection of ClO− have been reported
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and most of them were developed on the basis of strong oxidation properties of HClO.20ꢀ34 In
particular, fluorescein and its derivativesꢀbased fluorescent probes for ClOꢀ detection have
shown excellent photophysical properties， including good photostability， high fluorescence
quantum yields and so on.35ꢀ38 Hence, the conjugation of a HClOꢀrecognizing moiety, sensitive
to hypochlorite oxidation, with an organic fluorophore, including pꢀmethoxyphenol,39 thiol,40ꢀ45
selenide,46ꢀ48 dibenzoylhydrazine,49 hydroxamic acid50 and oxim derivatives51ꢀ53 have been
widely used for probe design. The C=N isomerization as a signaling mechanism have received
extensive attention since it was first designed as fluorescent chemosensor.54,55 The strong
fluorescence of organic fluorophore containing unbridged C=N bonds is known to undergo
nonꢀradiative decay process in the excited state, so those compounds are often nonfluorescent.56
After the unbridged C=N was removed by specific analytes through a chemical reaction, the
fluorescence intensity of compounds can be enhanced distinctly. Therefore, based on the C=N
isomerization and the oxidization hydrolysis of hydrazide, a sensitive and selective turn on
fluorescent probe can be established.
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The applications of most fluorescent probes are mainly focused on cells imaging. Besides,
accurate quantitative information about individual cells in a large cell population is extremely
important as well. To meet the demands, flow cytometry, as a powerful and widespread
technique, can not only be utilized for the measurement of cell phenotype and function but also
provide the accurate quantitative information of singleꢀcell among several millions of cells.57
Thus, it would be pretty attractive to apply flow cytometry to various biological studies and
clinical diagnoses. To date, there are a quite number of outstanding fluorescent probes for HClO
detection and imaging. However, a lack of sensitive and selective fluorescent probes for
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quantitative detection of HClO in cells has greatly hampered better understanding the role of
HClO in many biological vital processes. In this work, we report a sensitive and selective turn
on fluorescent probe with fast response time, for exogenous and endogenous imaging of
hypochlorous acid in living cells and quantitative application in flow cytometry.
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EXPERIMENTAL SECTION
Materials and instrumentations. All reagents were of analytical reagent grade and used
without further purification. 1H NMR, 13C NMR spectra were measured on a Bruker AM400
NMR spectrometer. Proton Chemical shifts of NMR spectra were given in ppm relative to
internals reference TMS (1H, 0.00 ppm). ESIꢀMS and HRMS spectral data were recorded on a
Finnigan LCQDECA and a Bruke Daltonics Bio TOF mass spectrometer, respectively.
Photoluminescence spectra were performed on a Hitachi Fꢀ7000 fluorescence spectrophotometer.
Unless otherwise noted, materials were obtained from commercial suppliers and were used
without further purification. Cells imaging was performed with Zeiss inverted fluorescence
microscope and flow cytometry experiment was performed in Beckman cytoflex.
Fluorescence analysis. Probe was dissolved in dimethylformamide (DMF) for a stock
solution (1 mM). Test solution of probe 1 (1×10ꢀ5 mol/L) was prepared in DMFꢀPBS solution (1:
9, v/v, 10 mM PBS, pH 7.4) and small aliquots of each testing species solution were added. The
resulting solutions were shaken well at room temperature before recording spectra.
Preparation of solutions of cations and anions and ROS and RNS. 1 mmol of inorganic
salt (NaCl, KCl, CaCl2·2H2O, CoCl2·6H2O, NiCl2·6H2O, CdCl2, CuCl2·2H2O, ZnCl2, PbCl2,
MgCl2·6H2O, MnCl2·4H2O, CrCl3·6H2O, FeCl3·6H2O, AlCl3·6H2O, NaF, KBr, KI, Na2S·9H2O,
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Na2SO4, Na2SO3, Na2S2O4, Na2S2O8, NaHSO3, Na2CO3, Na2C2O4, NaH2PO4·2H2O, NaNO2,
KSCN, NaHCO3, K2Cr2O7, NaClO3) and ascorbic acid (AA), glutathione (GSH), cysteine (Cys)
were dissolved in distilled water (100 mL) to afford 1×10ꢀ2 mol/L aqueous solution. The stock
solutions were diluted to desired concentrations with water when needed. Hypochlorite was
prepared by dilution of commercial NaClO solution in deionized water and assayed using a
spectrophotometer using ε292nm = 350 Mꢀ1cmꢀ1. Tertꢀbutyl hydroperoxide (TBHP) was prepared
by dilution of commercial TBHP solution. Hydrogen peroxide was prepared by dilution of
commercial H2O2 solution directly. Nitric oxide (NO) was prepared by treating sulfuric acid
solution with sodium nitrite solution and its stock solution was prepared by bubbling NO into
deoxygenated deionized water for 30 min. Peroxynitrite stock (ONOOꢀ) was prepared by
following literature procedure58 and the concentration of peroxynitrite was estimated by using a
spectrophotometer ε302nm = 1670 Mꢀ1cmꢀ1. Hydroxy radical was prepared by addition of Fe2+
solution into a solution containing excess H2O2 through Fenton chemistry. Superoxide was
generated from KO2 with a saturated solution of KO2 in DMSO.
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Synthesis of Probe 1. The synthesis of Probe 1 is shown in Scheme S1 in the Supporting
Information. The details are described as follows.
Synthesis of S1. Compound S1 was synthesized using a modified literature method.
Specifically,
A
solution of 1,10ꢀPhenanthrolineꢀ5,6ꢀdione (0.945 g, 4.5 mmol),
4ꢀHydroxybenzaldehyde (0.550 g, 4.5 mmol), aniline (0.50 mL, 5.5 mmol) and ammonium
acetate (3.48 g, 45 mmol) in glacial acetic acid (20 mL) was refluxed for 24 h under an argon
atmosphere, resulting in a colour change from darkꢀred to yellow. After being cooled to room
temperature, the reaction mixture was poured into ice water (400 mL), forming a yellow
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precipitate. With the addition of ammonium hydroxide, more precipitate formed, the resulting
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solid was collected by filtration, washed with water and acetone, and dried in vacuum to yield
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1
compound S1. Yield: 58%. HꢀNMR (400 MHz; DMSOꢀd6):
δ 9.90 (s, 1 Η), 9.06 (d, J = 2.40
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Hz, 1 H), 8.99 (d, J = 8.00 Hz, 1 H), 8.91 (d, J = 2.40 Hz, 1 H), 7.86 (dd, J = 7.60, 4.40 Hz, 1 H),
7.77ꢀ7.67 (m, 5 H), 7.42 (dd, J = 8.40, 4.40 Hz, 1 H), 7.42ꢀ7.37 (m, 2 H), 7.31 (d, J = 8.40, 1 H),
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6.75ꢀ6.70 ppm (m, 2H); CꢀNMR (100 MHz; DMSOꢀd6): δ 158.5, 152.3, 148.3, 147.2, 143.7,
143.5, 137.8, 135.0, 130.6, 130.5, 130.4, 129.7, 129.0, 127.1, 126.3, 123.7, 123.4, 122.4, 120.4,
119.4, 115.1 ppm; HRMS (ESI): calcd for C25H16N4O [M]ꢀ m/z =387.1246. Found: m/z =
387.1235.
Synthesis of S2. Compound S2 was synthesized using a modified Duff reaction. S1 (0.390 g,
1 mmol) and hexamethylene tetraamine (0.640 g, 4 mmol) were dissolved in 6 mL
trifluoroacetic acid and refluxed for three days. The mixture was allowed to cool to room
temperature and 30 mL of a 4M HCl solution were added and allowed to stir for 2 hours, during
which time the reaction became cloudy, the pH was adjusted to approximately three through the
addition of a saturated aqueous solution of NaHCO3, forming a yellow precipitate and the
resulting yellow solid was collected by filtration, washed with water, acetone, ether and dried in
vacuum to yield compound S2. Yield: 54%. 1HꢀNMR (400 MHz; DMSOꢀd6):
δ
11.30 (s, 1 Η), 10.24 (s, 1 H), 9.44 (d, J = 8.40 Hz, 1 H), 9.22 (d, J = 4.40 Hz, 1 H), 9.09 (d, J
= 4.40 Hz, 1 H), 8.23 (dd, J = 7.20, 4.80 Hz, 1 H), 7.87 (s, 1 H), 7.81ꢀ7.74 (m, 6 H), 7.73 (d, J =
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8.40 Hz, 1 H), 7.53 (d, J = 8.40 Hz, 1 H), 7.01 (d, J = 8.80 Hz, 1 H); CꢀNMR (100 MHz;
DMSOꢀd6): δ 189.6, 161.7, 157.9, 152.0, 146.7, 145.6, 138.2, 136.8, 136.2, 135.1, 134.5, 131.0,
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130.9, 129.7, 129.2, 128.7, 127.0, 125.4, 124.7, 124.1, 122.2, 120.21, 120.17, 117.5 ppm;
HRMS (ESI): calcd for C26H16N4O2 [M+H]+ m/z =417.1351. Found: m/z = 417.1358.
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Synthesis of S3. Compound S3 was synthesized using a literature method. To a suspension of
fluorescein (6.0 g, 18.1 mmol) in ethanol (24 mL) was added excess hydrazinium hydroxide (24
mL, 80% aqueous). The reaction mixture was heated to reflux and allowed to stir for 7 h, after
being cooled to room temperature, the reaction mixture was poured into ice water (400 mL) and
left to sit for 2 h. The resulting yellow precipitate was filtered from the aqueous suspension and
washed with water and cold ethanol. Recrystallisation of the precipitate in ethanol (250 mL) to
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yield the compound S3. Yield: 50%. 1H NMR (400 MHz, DMSOꢀd6)
δ 9.79 (s, 2 H), 7.81ꢀ7.77
(m, 1 H), 7.53ꢀ7.43 (m, 2 H), 7.03ꢀ6.95 (m, 1 H), 6.59 (s, 2 H), 6.46 (d, J = 8.80, 2 H), 6.41 (d, J
= 8.40 Hz, 2 H), 4.34 (s, 2 H). 13C NMR (100 MHz, DMSOꢀd6) 165.5, 158.2, 152.4, 151.5,
132.6, 129.3, 128.4, 127.9, 123.4, 122.3, 112.0, 109.9, 109.6, 102.3, 64.6 ppm; HRMS (ESI):
calcd for C20H14N2O4 [M]ꢀ m/z =345.0875. Found: m/z = 345.0862.
Synthesis of probe 1. Probe 1 was synthesized using a literature method. A solution of S2
(0.210 g, 0.5 mmol) and S3 (0.175 g, 0.5 mmol) in absolute ethanol (15 mL) was refluxed for 6
h. The solution was cooled, concentrated to 10 mL and allowed to stand at room temperature
overnight. The precipitate which appeared next day was filtered and washed 3 times with 10 mL
cold ethanol, recrystallisation of the crude product from EtOH to get pure product and dry in
vacuum to yield probe 1. Yield: 55%. 1HꢀNMR (400 MHz; DMSOꢀd6):
δ 10.77 (s, 1 Η), 9.99 (s,
2 H), 9.08 (d, J = 2.80 Hz, 1 H), 9.04 (d, J = 8.00 Hz, 1 H), 8.94 (d, J = 2.80 Hz, 1 H), 8.80 (s, 1
H), 7.96 (d, J = 7.20 Hz, 1 H), 7.90 (dd, J = 7.60, 4.40 Hz, 1 H), 7.75ꢀ7.57 (m, 8 H), 7.49 (dd, J
= 8.40 Hz, J = 4.00 Hz, 1 H), 7.34 (d, J = 8.40 Hz, 2 H), 7.13 (d, J = 7.60 Hz, 1 H), 6.76 (d, J =
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8.40 Hz, 1 H), 6.70 (s, 2 H), 6.56ꢀ6.47 (m, 4 H); CꢀNMR (100 MHz; DMSOꢀd6): δ 163.7.6,
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158.8, 157.8, 151.9, 151.3, 151.0, 148.3, 147.1, 147.3, 147.1, 143.5, 135.0, 134.3, 132.0, 130.5,
129.2, 128.8, 128.0, 127.9, 127.3, 126.4, 123.9, 123.7, 123.4, 122.6, 121.0, 119.4, 119.1, 116.3,
112.6, 109.6, 109.2, 102.7, 64.9 ppm; HRMS (ESI): calcd for C46H28N6O5 [M]ꢀ m/z =743.2043.
Found: m/z = 743.2012.
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Cell culture and imaging. HeLa cells and RAW264.7 cells were cultured in Dulbecco's
modified Eagle medium (DMEM) containing 10% fetal bovine serum and 1%
penicillinꢀstreptomycin at 37℃ in a 5% CO2/95% air incubator. For detection of exogenous
HClO, the cultured HeLa cells in a 6ꢀwell plate were washed three times with PBS and then
incubated with 20 ꢁM of probe 1 for 1.5 h at 37 °C in a 5% CO2/95% air incubator. After
washing three times with PBS, the cells were cultured for another 30 min with different
concentration of ClOꢀ. The cells were washed three times carefully with PBS and imaged. For
detection of endogenous HClO, RAW264.7 cells in 6ꢀwell plate were stimulated by LPS and
IFNꢀγfor 16 h in the culture medium and then PMA and probe 1 for another 2 h. The cells were
washed three times carefully with PBS and imaged.
Flow Cytometry Experiments. Cells were treated as described in Cell culture and imaging
part. Before measurement by flow cytometry, cells were scraped off gently and collected into a
clean 2 mL centrifuge tube. Then, cells were spun down (500 rpm, room temperature, 3 min).
After discarding the supernatant, 1 mL of warm PBS was added gently to reꢀsuspend the cell
pellet. Finally, cells were analyzed on a Beckman flow cytometer equipped with 488 nm Ar laser
and fluorescence was collected by PE channel.
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RESULTS AND DISCUSSION
Spectroscopic Response of Probe 1 to ClO-. Fluorescein was chosen as an ideal
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fluorescence reporting group due to its excellent photophysical properties and the structure of
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1
probe 1 was confirmed by H NMR, 13C NMR and ESIꢀTOF mass spectra (Fig S1). To
investigate the response of probe 1 to ClOꢀ, the fluorescence response behavior of probe 1
towards various concentrations ClOꢀ was measured at room temperature. As shown in Fig 1,
probe 1 exhibits very weak fluorescence due to both the spirolactam form of fluorescein and the
unbridged C=N bonds which can undergo nonꢀradiative decay process in the excited state. Upon
the addition of ClOꢀ, the fluorescence emission of probe 1 at 542 nm was greatly enhanced in
the range of 0ꢀ100 ꢁM. The reaction mechanism of probe 1 with ClOꢀ was proposed in Scheme
1 and the result was illustrated on the ESIꢀMS, 1H NMR and in suit FTꢀIR (Fig S7ꢀ S9).
To obtain efficient signaling conditions, various experimental conditions toward hypochlorite
were examined, including coꢀsolvent, buffer solution, pH and time. Firstly, we tested the
signaling behavior of probe 1 toward ClOꢀ in DMF and DMSO with different water percentages
(Fig S2 and Fig S3). It was found that compared with the signal enhancement in DMF, the signal
in DMSO was much lower due to the scavenging effect of DMSO for hypochlorite.59 probe 1
exhibited highest signal enhancement toward ClOꢀ in 10% DMFꢀPBS buffer solution at pH 7.4.
We were then make a comparison among three different buffer solutions, 10 mM PBS, 10 mM
TrisꢀHCl and 10 mM HEPES, to see whether the enhancement of fluorescence intensity can be
affected. Not surprisingly, the fluorescence responses were varied significantly quite different
when treated with ClOꢀ in Fig 2a. The fluorescence intensity was greatly enhanced in 10 mM
PBS buffer solution, slightly enhanced in 10 mM HEPES and almost remain constant in 10 mM
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TrisꢀHCl, demonstrating that partial ClOꢀ was consumed by TrisꢀHCl and HEPES. HEPES and
TrisꢀHCl can be oxidized by ClOꢀ and the oxidative activity of ClOꢀ was almost lost when ClOꢀ
was incubated with HEPES or TrisꢀHCl prior to probe 1 addition. Thus, HEPES and TrisꢀHCl
are inappropriate for ClOꢀ detection and the result is in good accordance with the literature.60,61
Subsequently, the fluorescence dynamics of probe 1 were investigated in 10% DMFꢀPBS
buffer (10 mM, pH 7.4) at room temperature. As shown in Fig. 2b, the reaction of probe 1 with
ClOꢀ was fast and the fluorescence intensity was significantly enhanced and remained stable
after 1 min, indicating that probe 1 is suitable for monitoring HClO in realꢀtime. Finally, the
effect of pH on the reaction was examined carefully. As shown in Fig 3a. Probe 1 showed
nearly no fluorescence at pH 5ꢀ10 and significant emission enhancement in the presence of ClOꢀ
over the investigated pH range of 7.4ꢀ10, indicating that probe 1 is suitable for imaging of ClOꢀ
in living cells.
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In order to evaluate the selectivity of probe 1 towards ClOꢀ, the influence of anions, cations
and various other biologically relevant analytes was investigated (Fig S4ꢀS6), including ascorbic
acid (AA), glutathione (GSH), cysteine (Cys), tertꢀbutyl hydroperoxide (TBHP), hydrogen
peroxide (H2O2), nitric oxide (NO), peroxynitrite anion (ONOOꢀ), hydroxyl radical (•OH), and
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superoxide (•O2 ). As shown in Fig 3b, there were no observable fluorescence enhancement
except with ClOꢀ. It is worth noting that the concentrations of those analytes were much higher
than ClOꢀ.
Fluorescence Imaging and flow cytometry quantitative detection of exogenous HClO in
HeLa Cells. To demonstrate the potential biological applications of probe 1, we utilized it for
the detection of intracellular HClO. Firstly, a MTT assay with HeLa cells and RAW264.7
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macrophages was examined to determine the cytotoxicity of probe 1. The results in Fig 4
showed that the viability of HeLa cells and RAW264.7 macrophages remained above 80% after
4 h and 24 h incubation with different concentrations of probe 1, indicating that probe 1 has
low cytotoxicity. Then HeLa cells were incubated with 20 ꢁM probe 1 for 1.5 h, very weak
fluorescence was observed (Fig S9). After the treatment of ClOꢀ (200 ꢁM) for 30 min, a stronger
green fluorescence was detected. Meanwhile, HeLa cells were also treated with various other
biologically relevant analytes. As expected in Fig S9, there were very weak fluorescence from
the cells and the result of flow cytometry experiment (Fig S10) was in accordance with imaging
experiment. The above result demonstrated that probe 1 has a turnꢀon fluorescence response and
high selectivity to HClO over various other biologically relevant analytes in living cells.
To evaluate the potential of visualizing HClO levels in living cells, the probe 1 loaded HeLa
cells were used for the imaging of different concentrations of exogenous HClO. HeLa cells were
coꢀincubated with 20 ꢁM probe 1 for 1.5 h. After the cells were incubated with different
concentrations of ClOꢀ (0 ꢁM, 50 ꢁM, 100 ꢁM, 200 ꢁM, 400 ꢁM) for another 30 min,
fluorescence images were then acquired. As shown in Fig 5. In the absence of ClOꢀ, the
fluorescence of cells is weak. In contrast, HeLa cells treated with 50 ꢁM ClOꢀ display obvious
fluorescence. Moreover, stronger fluorescence is observed when the cells were treated with a
higher concentration of ClOꢀ till 200 ꢁM. The ClOꢀ levels in Hela cells can also be qualitatively
acquired by flow cytometry. As can be seen in Fig 6a, the geometric mean of fluorescence
intensity was significantly enhanced with the increase of ClOꢀ concentration. The above
experiments proved that probe 1 has the ability to visualize and quantitatively detect ClOꢀ in
living cells.
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Fluorescence Imaging and flow cytometry quantitative detection of endogenous HClO in
RAW264.7 Cells. We then investigated whether the probe was capable of detecting LPS
(lipopolysaccharide)ꢀinduced endogenous produced HClO in RAW264.7 cells. It is known that
endogenous HClO can be produced under the stimulation of LPS, IFNꢀγ and phorbol myristate
acetate (PMA).62 Thus, RAW264.7 macrophages were coꢀincubated with various concentrations
of LPS (0.125 ꢁg/ml, 0.25 ꢁg/ml, 0.5 ꢁg/ml) and IFNꢀγ (50 ng/ml) for 16 h and then PMA (1
ꢁg/ml) and probe 1 (20 ꢁM) for 2 h. As shown in Fig 6b, the percentage of positive cells in flow
cytometry increased gradually with concentration of LPS increasing. 0.5 ꢁg/ml LPS was used
for stimulating RAW264.7 cells to produce H2O2. We further examined the applicability of
probe 1 for endogenous HClO imaging. As depicted in Fig 7a and 7b, the stimulated RAW264.7
cells displayed strong fluorescence, while almost no fluorescence emission was observed in
control group. Moreover, in another two groups, 4ꢀaminobenzoic acid hydrazide (ABH) was
added to decrease cellular HClO level through inhibiting the activity of MPO. The fluorescence
intensity can also be reduced clearly by Nꢀacetylcysteine (NAC), a powerful ROS scavenger. As
shown in Fig 7c and 7d, the fluorescence intensity is suppressed to some extent when
RAW264.7 cells were incubated with ABH and NAC, respectively. This result demonstrated that
endogenous HClO can be visualized with probe 1 in RAW264.7 cells.
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With the above encouraging results, we decided to detect endogenous HClO in flow
cytometry with probe 1. RAW264.7 macrophages were coꢀincubated with stimulus and probe 1
in the presence or absence of ABH or NAC. As shown in Fig 8a, only 0.3% positive cells can be
acquired in unstimulated group, whereas, in the presence of the stimulus, the percentage of
positive cells in flow cytometry can be significantly increased to 34.23% (Fig 8b). In addition,
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the percentage of positive cells was inhibited to 23.34% and 21.66% in the presence of ABH and
NAC respectively (Fig 8c and 8d). This result indicated that probe 1 have the ability to
quantitatively detect endogenous HClO in RAW264.7 cells by flow cytometry.
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CONCLUSIONS
In conclusion, we have successfully synthesized a turnꢀon fluorescent probe for highly
selective and sensitive detection of hypochlorous acid with fast response time and the probe can
be used for exogenous and endogenous imaging of hypochlorous acid in living cells.
Furthermore, probe 1 has shown superior ability for the quantitative detection of exogenous and
endogenous HClO in flow cytometry.
ACKNOELEDGEMENTS
We thank Dr. Pengchi Deng and Prof. Aiming Sun in Analytical & Testing Center of Sichuan
University for technical assistance. This work was supported by the National Natural Science
Foundation of China (Nos. 21375089 & 21505095) and Science & Technology Department of
Sichuan Province of China (2015JY0272).
Supporting Information Available: The Supporting Information is available free of charge on
the Internet at http://pubs.acs.org.
Chemical structures of compounds, NMR and MS spectra, additional spectral data, cells
imaging data and flow cytometry data.
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Figure 1. (a) Changes in the fluorescence spectra of probe 1 (10 ꢁM) with different
concentrations of NaClO (0ꢀ100 ꢁM). (b). Linear relationship between probe 1 and NaClO in
the concentration range of 0.5−100 ꢁM. λex/em = 480 / 542 nm
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Scheme 1. The structure of probe 1, and the proposed mechanism of fluorescence response to
ClOꢀ.
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Figure 2. (a) Fluorescence intensity of probe 1 (10 ꢁM) in 10 mM PBS, 10 mM TrisꢀHCl, 10
mM HEPES in the presence and absence of ClOꢀ at room temperature with excitation of 480 nm.
(b) Timeꢀdependent changes in fluorescence intensity of probe 1 (10 ꢁM) in the presence and
absence of ClOꢀ at room temperature with excitation of 480 nm
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Figure 3. (a) The effects of pH values on the fluorescence intensity in the presence and absence
of ClOꢀ at room temperature with excitation of 480 nm (b) Fluorescence spectra of probe 1
before and after reaction with various biologically relevant analytes AA (1 mM), GSH (1 mM),
Cys (1 mM), TBHP (200 ꢁM), H2O2 (1 mM), NO (200 ꢁM), ONOOꢀ (200 ꢁM), •OH (200 ꢁM),
•O2 (200 ꢁM). and ClOꢀ (200 ꢁM) in PBS (pH 7.4, containg 10% DMF) at room temperature
‑
with excitation of 480 nm.
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Figure 4. Effects of probe 1 at varied concentrations on the viability of HeLa cells and RAW264.7 cells.
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Figure 5. Fluorescence (upper) and bright field (lower) of HeLa cells treated with various
concentrations of ClOꢀ. (a) 0 ꢁM (b) 50 ꢁM (c) 100 ꢁM (d) 200 ꢁM (e) 400 ꢁM. Scale bar: 100
ꢁm.
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Figure 6. (a) Detection of exogenous ClOꢀ in flow cytometry with probe 1. HeLa cells were
coꢀincubated with probe 1 (20 ꢁM) and then treated with various concentrations of ClOꢀ for 30
min and analyzed by flow cytometry. (b) Detection of different concentrations of LPSꢀinduced
endogenous produced ClOꢀ in RAW264.7 with probe 1 in flow cytometry. Flow cytometer
equipped with 488 nm Ar laser and fluorescence was collected by PE channel.
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Figure 7. Fluorescence (upper) and bright field (lower) of RAW 264.7 cells treated with various
stimulants (a) probe 1 (b) LPS (0.5 ꢁg/ml), IFNꢀγ (50 ng/ml) for 16 h then PMA (1 ꢁg/ml) and
probe 1 (20 ꢁM) for 2 h (c) LPS (0.5 ꢁg/ml), IFNꢀγ (50 ng/ml), PMA (1 ꢁg/ml) and ABH (200
ꢁM) for 16 h then probe 1 (20 ꢁM) for 2 h (d) LPS (0.5 ꢁg/ml), IFNꢀγ (50 ng/ml), PMA (1
ꢁg/ml) and NAC (1 mM) for 16 h then probe 1 (20 ꢁM) for 2 h. Scale bar: 200 ꢁm.
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Figure 8. Detection of endogenous ClOꢀ in flow cytometry with probe 1 (a) probe 1 (b) LPS
(0.5 ꢁg/ml), IFNꢀγ (50 ng/ml) for 16 h then PMA (1 ꢁg/ml) and probe 1 (20 ꢁM) for 2 h (c) LPS
(0.5 ꢁg/ml), IFNꢀγ (50 ng/ml), PMA (1 ꢁg/ml) and ABH (200 ꢁM) for 16 h then probe 1 (20
ꢁM) for 2 h (d) LPS (0.5 ꢁg/ml), IFNꢀγ (50 ng/ml), PMA (1 ꢁg/ml) and NAC (1 mM) for 16 h
then probe 1 (20 ꢁM) for 2 h
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