A two-photon ESIPT based fluorescence probe for specific detection of hypochlorite

Article abstract of DOI:10.1016/j.dyepig.2018.01.027

Hypochlorous acid (HOCl) is one important member of reactive oxygen species (ROS) familyresponsible for various human diseases. Although many contributions have been made to develop fluorescent probes for sensing HOCl, many of them suffer small Stokes shifts, sophisticated synthetic procedure, and slow reaction rate. In the current study, we designed and synthesized an ESIPT based fluorescence probe with diaminomeleonitrile (DMN) as reactive site. The probe can detect HOCl with high selectivity and sensitivity. It also has large Stokes shift and rapid reaction rate. Moreover, it can be utilized to monitor exogenous and endogenous HOCl in living cells and tissues via two-photon microscopy.

Full text of DOI:10.1016/j.dyepig.2018.01.027

Dyes and Pigments xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
A two-photon ESIPT based fluorescence probe for specific detection of
hypochlorite
Liyan Chena,1, Sang Jun Parkb,1, Di Wu , Hwan Myung Kimb,∗∗, Juyoung Yoona,∗
a
a
Department of Chemistry and Nano Science, Ewha Womans University, Seoul, 120-750, South Korea
Department of Chemistry and Energy Systems Research, Ajou University, Suwon, South Korea
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
ESIPT
Hypochlorite
Two-photon
Living cells
Tissue imaging
Hypochlorous acid (HOCl) is one important member of reactive oxygen species (ROS) familyresponsible for
various human diseases. Although many contributions have been made to develop fluorescent probes for sensing
HOCl, many of them suffer small Stokes shifts, sophisticated synthetic procedure, and slow reaction rate. In the
current study, we designed and synthesized an ESIPT based fluorescence probe with diaminomeleonitrile (DMN)
as reactive site. The probe can detect HOCl with high selectivity and sensitivity. It also has large Stokes shift and
rapid reaction rate. Moreover, it can be utilized to monitor exogenous and endogenous HOCl in living cells and
tissues via two-photon microscopy.
1. Introduction
and serious self-quenching due to excitation backscattering effects.
Moreover, many probes suffer one limitation that they are always
Reactive oxygen species (ROS) and nitrogen species (RNS) including
hydrogen peroxide (H ), hydroxyl radical (∙OH), hypochlorous acid
evaluated by relatively short excitation wavelength which can easily
lead to autofluorescence, photodamage, and shallow tissue penetration
depths. In contrast, two-photon microscopy (TPM) can excite molecule
utilizing two near-infrared photons. It possesses various advantages
including minimized background emission, prolonged observation
time, and increased penetration depths [67–71]. Therefore, TPM has
become a meaningful and valuable approach for monitoring HOCl in
living cells and tissues. In this study, we developed a new fluorescence
probe based on familiar excited state intramolecular proton transfer
(ESIPT) fluorophore with large Stokes shifts and easy synthetic
pathway. To further demonstrate its biological value, the probe was
successfully used to sense exogenous and endogenous HOCl in living
cells and tissues via TPM.
2 2
O
−
1
(
2 2
HOCl), superoxide (∙O ), singlet oxygen ( O ), nitric oxide (NO), and
−
peroxynitrite (ONOO ) are involved in a number of physiological and
pathological processes [1–5]. Being one biologically important member
of ROS family, HOCl is produced from peroxidation of chloride ions
−
(
2 2
Cl ) and hydrogen peroxide (H O ) under catalysis of myeloperox-
idase (MPO) which is a potent oxidant and antimicrobial agent for the
immune system [6–8]. However, abnormal production of HOCl could
lead to various human diseases such as cardiovascular and kidney
diseases, inflammatory disease, neuron degeneration, and cancer
[
9–11]. Thus, it is urgently required to develop reliable methods to
monitor concentrations of HOCl in order to further investigate phy-
siological functions of HOCl in living systems. In contrast to various
traditional analytical techniques for detecting HOCl including electro-
analysis, potentiometry, chemiluminescence, and chromatography
2. Experimental section
[
12–15], fluorescence probes possess a number of advantages such as
2.1. General methods
high selectivity and sensitivity with rapid response rate and capability
of real-time detection [16–28]. In this regard, numerous fluorescence
probes for monitoring HOCl have been developed in the past few years
Unless otherwise noted, materials were obtained from commercial
1
suppliers and used without further purification. H NMR spectra were
[
29–66]. Unfortunately, some fluorescence probes need tedious syn-
recorded at 300 MHz and 13C NMR spectra were recorded at 75/
125 MHz. Chemical shifts were reported using δ scale in parts per
million (ppm) relative to the singlet (0 ppm) for tetramethylsilane
thetic procedures. Some of them show delayed response time. Some of
them also possess small Stokes shifts which may result in sensing error
∗
Corresponding author.
Corresponding author.
∗
∗
E-mail addresses: kimhm@ajou.ac.kr (H.M. Kim), jyoon@ewha.ac.kr (J. Yoon).
Liyan Chen and Sang Jun Park contributed equally to this work.
1
https://doi.org/10.1016/j.dyepig.2018.01.027
Received 4 January 2018; Received in revised form 17 January 2018; Accepted 18 January 2018
0143-7208/ © 2018 Elsevier Ltd. All rights reserved.
Please cite this article as: Chen, L., Dyes and Pigments (2018), https://doi.org/10.1016/j.dyepig.2018.01.027

L. Chen et al.
Dyes and Pigments xxx (xxxx) xxx–xxx
(
(
TMS). Data are reported as follows: chemical shift, multiplicity
s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of
measured at 710–880 nm using rhodamine 6G as reference with well-
characterized two-photon property in the literature [74]. Intensities of
the two-photon induced fluorescence spectra of the reference and
sample emitted at the same excitation wavelength were then de-
termined. TPA cross section was calculated using the following formula:
doublets, m = multiplet), coupling constants (Hz) and integration.
Fluorescence emission spectra were obtained using a RF-5301/PC
spectrofluorophotometer (Shimadzu). UV absorption spectroscopy
measurements were carried out on a Scinco S-3100 using a 1-cm optical
path length cell at room temperature. Confocal microscopy was con-
ducted with a confocal laser scanning biological microscope (Olympus,
FV1200, Japan) with 559 nm diode laser as excitation source.
r s r r r r s r r
δ = δ (S Ф ϕ c )/(S Ф ϕ c ) where subscripts s and r were sample and
reference molecules, respectively. The intensity of the signal collected
by a CCD detector was denoted as S. Φ was the fluorescence quantum
yield. ϕwas the overall fluorescence collection efficiency of the ex-
perimental apparatus. The number density of the molecule in solution
r
was denoted as c. δ was the TPA cross section of the reference molecule.
2.2. Synthesis of probe 1
Compound4 was synthesized with 61% yield using the following
2.5. Cell imaging study
procedure according to the published procedure [72]: First, 2-amino-
fenol2 (328 mg, 3.0 mmol) and salicylic acid3 (415 mg, 3.0 mmol) were
dissolved in polyphosphoric acid (PPA, 10 mL). The reaction was then
heated to 180 °C and stirred overnight. The mixture was cooled down
and poured into ice-cold water. The precipitate was then filtered fol-
lowed by washing with water and cold MeOH. A yellow solid was then
used for the next step directly.
Compound 5 was synthesized with 57% yield using published pro-
cedure [72]. Briefly, synthesized compound 4 (317 mg, 1.5 mmol) and
hexamethylenetetramine (HMTA, 350 mg, 2.5 mmol) were dissolved in
All cells were passed and plated onto glass-bottomed dishes (NEST)
before imaging for two days. They were maintained in a humidified
atmosphere of 5/95 (v/v) of CO
medium was removed and replaced with serum-free medium. These
cells were then treated with 10 μM probe at 37 °C under 5% CO for
2
/air at 37 °C. For labeling, the growth
2
30 min. The culture medium for Raw 264.7 cells (ATCC, Manassas, VA,
USA) was DMEM (WelGeneInc, Seoul, Korea) supplemented with 10%
FBS (WelGene), penicillin (100 units/ml), and streptomycin (100 μg/
mL).
10 mL polyphosphoric acid in a round-bottom flask. The reaction was
then heated to 100 °C and stirred overnight until most of compound 4
was consumed. This was monitored by TLC. The reaction was cooled
down and 30 mL of ice-cold water was added, leading to a white pre-
cipitate. The solid was purified by column chromatography with di-
chloromethane to afford compound 5.
2
.6. Preparation and staining of fresh rat hippocampal slices
Rat hippocampal slices were prepared from the hippocampi of 2-
weeks-old rat (SD) according to an approved institutional review board
protocol. Coronal slices were cut into slices with thickness of 400 μM
using a vibrating-blade microtome in artificial cerebrospinal fluid
Probe 1 was synthesized with 64% yield using the following pro-
cedure. Compound 5 (120 mg, 0.5 mmol) and diaminomaleonitrile
(
ACSF; 138.6 mM NaCl, 3.5 mM KCl, 21 mM NaHCO 0.6 mM
3
,
(
162 mg, 1.5 mmol) were dissolved in anhydrous EtOH. The reaction
NaH PO , 9.9 mM D-glucose, 1 mM CaCl , and 3 mM MgCl ). Slices
2
4
2
2
was heated to reflux and stirred overnight. The reaction was then
cooled down to room temperature until white solids precipitated from
the viscous solution. The solid was filtered and washed with cold
MeOH.
were incubated with 100 μM probe in ACSF bubbled with 95% O
2
and
5
% CO for 1.5 h at 37 °C. Slices were then washed three times with
2
ACSF and transferred to glass-bottomed dishes (NEST). They were then
observed with a spectral confocal multiphoton microscope.
2.3. Determination of fluorescence quantum yield
3. Results and discussion
The fluorescence quantum yields of probe 1 and product 1-ClO were
3.1. Probe design and synthesis
determined with quinine sulfate (Φ = 0.58, in 1 N H
2 4
SO ) as the re-
ference. The quantum yields were calculated using the following
As important members of ESIPT fluorophores, 2-(2′-hydroxyphenyl)
2
2
equation: Φ
absorbance of quinine sulfate and sample solutions at the same ex-
citation wavelength; F and F are the corresponding integrated fluor-
escence intensities of quinine sulfate and sample solutions; n and n are
the refractive index of 1N H SO and water.
x s s x x s x s x s
= Φ [(A F /A F ) (n /n )]. Where, A and A are the
benzoxazole (HBO) derivatives have been widely used in fluorescence
probes because they usually undergo large Stoke shifts [75–89].
Moreover, HBOderivatives possess two photon (TP) cross-sections with
capabilities for two-photon excitation fluorescence (TPEF) imaging.
Thus, in the current study, a new HBO based probe 1 was designed
utilizing diaminomeleonitrile (DMN) as the reactive site. We proposed
that HOCl could induce transformation of –C=N- group to –CHO,
leading to obviously fluorescent changes. In order to confirm this pro-
posal, probe 1 was synthesized through a simple three-step sequence
(Scheme 1). Initially, intermediate 4 was synthesized utilizing com-
mercially available 2-aminofenol 2 and salicylic acid 3. Intermediate 5
was then obtained by introducing aldehyde moiety to the para-position
x
s
s
x
2
4
2.4. Measurement of two-photon cross section
Two-photon cross section (δ) was determined using femtosecond
fs) fluorescence measurement technique as described previously [73].
Probe 1 (1.0 × 10 M) was dissolved in PBS buffer (10 mM, pH 7.4,
containing 20% DMF). Two-photon induced fluorescence intensity was
(
−
6
Scheme 1. The synthetic pathway of probe 1.
2

L. Chen et al.
Dyes and Pigments xxx (xxxx) xxx–xxx
Fig. 2. a) Fluorescence spectra of probe 1 (10 μM)before and after addition of 200 μM
−
1
−
•-
2 2 2 2 2
different analytes (H O , NO, ONOO , ROO, O , •OH, NO , O , HOCl) in PBS buffer
Fig. 1. a) Fluorescence spectra of probe 1 (10 μM) upon the addition of HOCl solution
(1.0 mM, pH = 7.4) with 20% DMF as co-solvent,λex = 360 nm, Slits: 5/5 nm; b)
Fluorescence intensity at 435 nm of probe 1 (10 μM) before and after addition of200 μM
different analytes in PBS buffer (1.0 mM, pH = 7.4); Spectra were recorded after in-
cubation with different analytes for 10min.
(
0–180 μM) in PBS buffer (1.0 mM, pH = 7.4) with 20% DMF as the co-solvent; b)
Fluorescence response of probe 1 at 435 nm to HOCl concentration (0–180 μM); Spectra
were recorded after incubation with different concentrations of HOCl for 15 min; Spectra
were recorded after incubation with different concentrations of HOCl for 10 min;
λ
ex = 360 nm; Slits: 5/5 nm.
a slight enhancement of the absorption band centered at 350 nm was
observed (ε350 = 1.67 × 10 ) (Fig. S2).
4
of hydroxyl group. Probe 1 was finally obtained through reaction of
intermediate 5 and diaminomeleonitrile (DMN) (Scheme 1 and Scheme
S1). Among these three steps, only the preparation of intermediate 5
needed to be purified by column chromatography, while both 4 and
probe 1 could be purely obtained by simple filtrations.
3.3. Selectivity of probe 1 toward HOCl
In order to investigate the specificity of probe 1 toward HOCl,
fluorescent responses of probe 1 toward other reactive species were
further monitored in PBS buffer (1.0 mM, pH = 7.4). Results are shown
in Fig. 2. Only the addition of HOCl resulted in remarkable enhance-
ment of fluorescence intensity at 435 nm. However, other reactive
3
.2. Spectral properties of probe 1 towards HOCl
−
−
species including H
2
O
2
, NO, ONOO , ROO, 1
O
2
, •OH, NO
2
, and
With the probe in hand, the ability of probe 1to detect HOCl in
•
−
O
2
failed to induce obvious fluorescent changes even under high
solutions was investigated first. The free probe 1 was nearly non-
concentrations. The histogram in Fig. 2b displayed that fluorescence
enhancement caused by HOCl was much higher than that by other re-
levant analytes, indicating the high selectivity of probe 1 toward HOCl.
fluorescent (Φ = 0.06) due to strong photoinduced electron transfer
(
PET) process of the amine group in DMN when it was dispersed in PBS
buffer (1.0 mM, pH = 7.4). Upon gradual addition of HOCl, a sig-
nificant enhancement of fluorescence intensity at 440 nm (Φ = 0.65)
with more than 80 nm Stokes shift was observed (Fig. 1a). Moreover, as
shown in Fig. 1b, the intensity of fluorescence at 435 nm showed a good
linear response to the concentration of HOCl. The detection limit of
probe 1 for HOCl was calculated to be 0.08 μMon the basis of 3δ/k,
displaying a high sensitivity toward HOClin aqueous solutions (Fig. S1).
3.4. Kinetic and mechanism study
Rapid reaction rate is another important condition for a qualified
fluorescence probe. Thus, time-based fluorescence changes of probe 1
and HOCl were assessed. As shown in Fig. 3, when five equivalents of
HOCl were added to the probe solution, a dramatic enhancement of
fluorescence intensity was rapidly observed at 435 nm. The maximum
Additionally, the free probe 1 displays an absorption band at 350 nm
4
(
ε
350 = 1.49 × 10 ). With the additions of increasing amounts of HOCl,
3

L. Chen et al.
Dyes and Pigments xxx (xxxx) xxx–xxx
2 2
myristate acetate (PMA)with the ability to produce H O which was
subsequently transformed to HOCl under catalysis of MPO. As shown in
Fig. 4c, after LPS/PMA-pretreated cells were incubated with probe 1 for
30 min, a notable enhancement in two-photon excited fluorescence
occurred. Moreover, when cells were treated with 4-amino-benzoicacid
hydrazide (ABAH) or flufenamic acid (FFA) (well-known MPO in-
hibitors) together with LPS/PMA followed by incubation with probe 1
for 30 min, the intensity of fluorescence was distinctly suppressed under
TPM. These results demonstrate that probe 1 possesses capabilities of
sensing exogenous and endogenous HOCl in living cells via TPM ima-
ging.
3.6. Tissue imaging of probe 1
In order to further explore the potential of probe 1for biological
applications, the ability of probe 1 to detect HOCl in living tissues was
further demonstrated. Rat hippocampal slices were prepared from the
hippocampi of 2-weeks-old rat (SD) and then incubated with 100 μM
probe 1 and 100 μM 1-ClO at 37 °C, respectively. From images shown in
Fig. 5, slices incubated by probe 1 displayed faint fluorescence (Fig. 5a).
However, when slices were incubated with 1-ClO, strong fluorescence
was observed in the CA1 and CA3 regions. Moreover, after slices were
activated with PMA, TPEF intensity was remarkably enhanced. Average
PMA-induced fluorescence intensities were between probe 1 and 1-ClO
labeled slices. Taken together, these results demonstrate that probe 1
possesses the capability of sensing endogenous HOCl in living tissues
via TPM.
Fig. 3. Time-dependence detection of fluorescent intensity at 435 nm for solutions of
probe 1 (10 μM) followed by addition of HOCl (50 μM or 80 μM), λex = 360 nm, Slits: 5/
5
nm.
fluorescence intensity was achieved within 30 s. If higher concentra-
tions of HOCl were added, less time was required to reach the max-
imum fluorescence intensity. The pseudo-first-order rate constant (kobs
for the reaction of probe and HOCl was calculated to be
based on kinetic data (Fig. S3).
According to the sensing mechanism reported previously [55–65], it
)
1
−
1
−1
1
.4 × 10 sec
is believed that reactions of HOCl and probe 1 couldpromote the con-
version of imine -CH=N- group to -CHO group (Scheme 1 and Fig. S4).
The generated aldehyde containing product1-ClO is responsible for the
increased fluorescence intensity owing to the inhibition of PET process.
In order to further confirm the proposed mechanism (Scheme 2 and Fig.
S4), probe 1 (10 μΜ) and HOCl (10 μΜ) was mixed with PBS buffer. It
was then monitored under high-resolution mass spectrometry (HRMS)
4
. Conclusions
In summary, a two-photon fluorescence probe for detecting HOCl
was successfully developed based on familiar ESIPT fluorophore HBO.
This probe displayed high selectivity for sensing HOCl over other re-
levant reactive species, including H
NO
tecting HOCl with high sensitivity, rapid response rate, and large Stokes
shift. Moreover, the biologically practical value of this probe was de-
monstrated by the fact that it could be utilized to monitor exogenous
and endogenous HOCl in the living cells and tissues under two-photon
microscopy.
−
1
2
O
2
, NO, ONOO , ROO,
2
O , •OH,
(
Fig. S4). Anew main peak appeared at 240.0671 in the mass spectrum.
−
•-
2 2
, and O . Furthermore, this probe possessed capabilities of de-
This matched with the calculated mass of 240.0661 for 1-ClO ([M
+
+
3
H] : (C14H10NO ).
3.5. Two-photon cell imaging of probe 1
Inspired by the successful use of probe 1 for sensing HOCl in solu-
tions, utilizations of probe 1 to detect exogenous and endogenous HOCl
in living cells were then evaluated. Due to various advantages such as
low background emission and minimized photodamage of two-photon
microscopy (TPM), we attempted to evaluate the utility in RAW 264.7
macrophages under two-photon mode. TPM fluorescence images were
initially obtained after cells were treated with 200 μM HOCl for 30 min
followed by incubation with probe 1. As shown in Fig. 4, remarkable
fluorescence enhancement was observed in contrast to faint fluores-
cence emitted by free probe 1. Such promising results indicated that
probe 1 had the capability to sense exogenous HOCl in living cells. This
further inspired us to investigate the feasibility of using probe 1 to
monitor endogenous HOCl. According to a previous report [41],
RAW264.7 macrophage cells were firstly stimulated by lipopoly-
saccharide (LPS) and IFN-γ. They were then incubated with phorbol
Acknowledgments
This study was supported by grants of the National Research
Foundation of Korea (NRF) funded by the Korean government (MSIP)
(
2012R1A3A2048814 to J. Y.; 2016R1E1A1A02920873 to H. M. K)
Mass spectral data were ob-tained from the Korea Basic Science
Institute (Daegu) using a Jeol JMS 700 high-resolution mass spectro-
meter.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.
doi.org/10.1016/j.dyepig.2018.01.027.
Scheme 2. Reaction mechanism of probe 1 and HOCl.
4

L. Chen et al.
Dyes and Pigments xxx (xxxx) xxx–xxx
Fig. 4. TPM images of RAW 264.7 cells incubated
with (a-e)probe 1. (a) Control image; (b) Cells
pretreated with NaOCl (200 μM) for 30 min and
then incubated with probe 1 (10 μM)for 30 min;
(
c) Cells pretreated with LPS (100 ng/mL) for
1
3
6 h, IFN- γ (50 ng/mL) for 4 h, PMA (10 nM) for
0 min, and then incubated with probe 1;(d) Cells
pretreated with LPS for 16 h, IFN-γ for 4 h, PMA
10 nM) + 4-ABAH (50 μM) for 4 h, and then
(
incubated with probe 1; (e) Cells pretreated with
LPS for 16 h, IFN-γ for 4 h, PMA (10 nM) + FFA
(
(
50 μM) for 4 h, and then incubated with probe 1;
f) Average TPEF intensity in (a-e); Images were
acquired using 740 nm excitation and emission
windows of 400–600 nm. Scale bars: 20 μm in a
and b; 25 μm in c, d and e.
Fig. 5. Tissue images of a rat hippocampal slice stained with
probe 1 and 1-ClO. TPM images of a rat hippocampal slice
stained with (a) probe 1 (100 μM) and (c) 1-ClO (100 μM); (b)
Tissue pretreated with PMA (10 ng/mL) for 30 min before la-
beling with probe 1; (d) Bright-field image of CA1 and CA3 re-
gions at 10x magnification; (e) Average TPEF intensities in (a-c);
TPM images were acquired using 740 nm excitation and emis-
sion windows of 400–600 nm in the neuron layer of CA3 regions.
Scale bars: 25 μm.
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