A highly selective two-photon probe with large turn-on signal for imaging endogenous HOCl in living cells

Article abstract of DOI:10.1016/j.dyepig.2017.07.006

Hypochlorous acid (HOCl) is one of the typical reactive oxygen species (ROS), which plays a crucial role in the immune system, and is involved in many neurodegenerative disorders, such as ischemic stroke, Alzheimer's, and Parkinson's disease. A two-photon

Full text of DOI:10.1016/j.dyepig.2017.07.006

Accepted Manuscript
A highly selective two-photon probe with large turn-on signal for imaging endogenous
HOCl in living cells
Jingjing Cao, Dong-Ming Jiang, Xin Ren, Ting Li, Xiao-Ting Gong, Yong-Rui Yang,
Zhu-Guo Xu, Chun-Lin Sun, Zi-Fa Shi, Shengxiang Zhang, Hao-Li Zhang
PII:
S0143-7208(17)30980-4
10.1016/j.dyepig.2017.07.006
DYPI 6095
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 28 April 2017
Revised Date: 30 June 2017
Accepted Date: 2 July 2017
Please cite this article as: Cao J, Jiang D-M, Ren X, Li T, Gong X-T, Yang Y-R, Xu Z-G, Sun C-L, Shi
Z-F, Zhang S, Zhang H-L, A highly selective two-photon probe with large turn-on signal for imaging
endogenous HOCl in living cells, Dyes and Pigments (2017), doi: 10.1016/j.dyepig.2017.07.006.
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ACCEPTED MANUSCRIPT
A Highly Selective Two-Photon Probe with Large Turn-On
Signal for Imaging Endogenous HOCl in Living Cells
a
a
b
b
a
Jingjing Cao , Dong-Ming Jiang , Xin Ren , Ting Li , Xiao-Ting Gong ,
Yong-Rui Yang a, Zhu-Guo Xu a, Chun-Lin Sun a, Zi-Fa Shi a, Shengxiang Zhang
b, * and Hao-Li Zhang a, *
a State Key Laboratory of Applied Organic Chemistry (SKLAOC); Key Laboratory of
Special Function Materials and Structure Design (MOE); College of Chemistry and
Chemical Engineering, Lanzhou University, Lanzhou, 730000, P. R. China
b School of Life Sciences, Lanzhou University, Lanzhou 730000, P. R. China
Tel: +86-0931-8912365 (H.Z.)
E-mail: haoli.zhang@lzu.edu.cn (H.Z.), sxzhang@lzu.edu.cn (S.Z.)

ACCEPTED MANUSCRIPT
A Highly Selective Two-Photon Probe with Large Turn-On
Signal for Imaging Endogenous HOCl in Living Cells
a
a
b
b
a
Jingjing Cao , Dong-Ming Jiang , Xin Ren , Ting Li , Xiao-Ting Gong ,
Yong-Rui Yang a, Zhu-Guo Xu a, Chun-Lin Sun a, Zi-Fa Shi a, Shengxiang Zhang
b, * and Hao-Li Zhang a, *
a State Key Laboratory of Applied Organic Chemistry (SKLAOC); Key Laboratory of
Special Function Materials and Structure Design (MOE); College of Chemistry and
Chemical Engineering, Lanzhou University, Lanzhou, 730000, P. R. China
b School of Life Sciences, Lanzhou University, Lanzhou 730000, P. R. China
Tel: +86-0931-8912365 (H.Z.)
E-mail: haoli.zhang@lzu.edu.cn (H.Z.), sxzhang@lzu.edu.cn (S.Z.)
Abstract
Hypochlorous acid (HOCl) is one of the typical reactive oxygen species (ROS), which
plays a crucial role in the immune system, and is involved in many neurodegenerative
disorders, such as ischemic stroke, Alzheimer's, and Parkinson's disease. A
two-photon probe for sensing HOCl with high selectivity, fast response (within 30 s),
good penetration depth and low cytotoxicity is reported. Under two-photon excitation,
the probe exhibited a remarkable fluorescence ‘‘turn-on’’ response to HOCl.
Furthermore, the probe was successfully applied to the monitoring of endogenous
HOCl stimulated by lipopolysaccharide in living microglia BV-2 cells.
Keywords: Two-photon fluorescent probe; Reactive oxygen species; Hypochlorous
acid; Endogenous bio-imaging
1. Introduction
Reactive oxygen species (ROS) have recently received increasing attention due to
their essential roles in various physiological and pathological processes [1-3], such as
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anti-inflammatory regulation, pathogen response, cellular signaling, and aging [4-7].
Among the various ROS, hypochlorous acid (HOCl) generated from myeloperoxidase
(MPO)-catalyzed peroxidation of chloride anions is a powerful antimicrobial agent
contributing much in human immune defense system [8-10]. However, mounting
evidence recently indicates that excessive accumulation of HOCl in living systems is
implicated in neurodegenerative disorders [11], such as ischemic stroke , Alzheimer's,
and Parkinson's disease [12-14]. Thus, it is highly desired to develop a fast and
efficient method for monitoring HOCl in the presence of H2O2 and other ROS for an
in-depth understanding of HOCl action mechanism in living organisms.
To date, many efforts have been made to develop HOCl fluorescent probes with
different recognition moieties including oxime [15], thioester [16,17], dibenzoyl
hydrazide [18], selenide [19,20] and other groups [21-23]; some of these probes have
been successfully implemented in bio-imaging. However, many probes were based on
conventional one-photon microscopy (OPM) and suffered shallow penetration depth
limiting their biological applications in vivo and in vitro. Two-photon microscopy
(TPM) is emerging as an effective tool for imaging in deep tissues due to its favorable
properties, such as deep penetration, reduced photo-damage and photo-bleaching,
lower background fluorescence and high observing resolution [24-27]. Unfortunately,
two-photon absorption materials with large two-photon absorption action
cross-section (Φδ), a key factor representing their brightness under illumination,
usually possess extended conjugated planar structures [28], which limited their cell
permeability. Most of previously reported small molecule two-photon probes based on
classical one-photon dyes exhibited high cell permeability but modest or small Φδ
values, such as rhodamine, fluorescein, and coumarin [29]. Consequently, it is
valuable to develop small molecule two-photon fluorescent probes with large
two-photon action cross-section (above 1000 GM) for bio-imaging applications.
In this work, a two-photon probe OPV-MEP (Scheme 1) bearing two oxathiolane
groups as the HOCl recognition sites and oligo (p-phenylene vinylene) (OPV)
skeleton as the two-photon chromophore was synthesized. The oxidative deprotection
of OPV-MEP by HOCl produces a compound OPV-CHO with large two-photon
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absorption action cross-section (1131.5 GM). The OPV-MEP displays a fast and
selective fluorescent turn-on response towards HOCl on two-photon excitation.
Moreover, the probe was successfully utilized in two-photon imaging of endogenous
HOCl in activated microglia BV-2 cells which can produce abundant HOCl in the
cytoplasm. The cytotoxicity of the probe was also investigated.
2. Experimental Section
2.1. General information
All reagents were purchased without further purification and solvents were dried
1
according to standard procedures before use. H-NMR and 13C-NMR spectra were
performed on an INOVA-600 MHz instrument (Agilent) with TMS as internal
standard. HRMS spectra were performed on a LTQ-Obitrap-ETD spectrometer.
Absorption spectra were recorded on a T6 UV-vis spectrophotometer (Purkinje
General). Fluorescence spectra were obtained on a FLS920 fluorimeter (Edinburgh
1
Instruments). Various ROS including HOCl, ·OH, O2, H2O2, ONOO-, NO, TBO·,
TBHP were prepared according to the procedure reported previously [30].
2.2. Synthesis of the probe
2.2.1.
Synthesis
of
tetraethyl((2,5-dimethoxy-1,4-phenylene)bis(methylene))bis(phosphonate) (2)
1,4-Dimethoxybenzene (1.38 g, 10 mmol) was dissolved in 1,4-dioxane (250 mL)
contained in the three-necked flask, then the solution was added with aqueous
formaldehyde solution (35%, 5.8 g, 68 mmol). The reaction was processed by the
gradual inlet of hydrogen chloride gas into the reactor at room temperature for 4 hours
until the reaction was completed. Saturated sodium bicarbonate solution was added to
the reaction mixture, and the product was extracted with dichloromethane. The
organic layer was dried with anhydrous Na2SO4, followed by evaporation of the
solvent. The crude product 1 was obtained as colourless crystals in 90% yield. MP:
1
155.8-157.2 °C. H-NMR (600 MHz, CDCl3), δ (ppm): 6.93 (s, 2H), 4.64 (s, 4H),
13
3.86 (s, 6H). C-NMR (150 MHz, CDCl3), δ (ppm): 151.16, 126.89, 113.42, 56.28,
+
41.26. HRMS (ESI): m/z calcd for C10H12Cl2O2 [M]+ 234.0209, found: 234.0211. IR
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(KBr, cm-1): 3437(w), 2968(m), 1738(w), 1510(s), 1462(s), 1410(s), 1320(m), 1223(s),
1037(s), 876(m), 737(m), 681(s), 608(m), 472(w).
Triethyl phosphite (29.13 g, 160 mmol) was added to the foregoing 1 (1.88 g, 8.0
mmol) contained within the flask and the solution was heated to 80 °C for 2 h. Then
the crude product was purified by recrystallization with chloroform/hexane to afford 2
1
as colourless plate-like crystals in 86% yield. MP: 127.3-129.2 °C. H-NMR (600
MHz, CDCl3), δ (ppm): 6.91 (s, 2H), 4.05-4.00 (m, 8H), 3.80 (s, 6H), 3.22 (d, J =
20.4 Hz, 4H), 1.26-1.23 (m, 12H). 13C-NMR (150 MHz, CDCl3), δ (ppm): 151.00,
1
119.41, 114.04, 61.81, 56.10, 26.44 (d, JCP = 139.21 Hz), 16.27. HRMS (ESI): m/z
+
calcd for C18H34O8P2 [M+H]+ 439.1645, found: 439.1638. IR (KBr, cm-1): 2985(s),
2839(m), 1520(s), 1414(m), 1271(s), 1215(s), 1049(s), 962(s), 883(m), 820(m),
631(m), 519(m).
2.2.2. Synthesis of 2,2'-(((1E,1'E)-(2,5-dimethoxy-1,4-phenylene)bis(ethene-2,1-diyl))
bis(4,1-phenylene))bis(1,3-oxathiolane) (OPV-MEP)
1,4-Phthalaldehyde (610 mg, 5.0 mmol) was dissolved in benzene (25 mL) in a
two-necked flask, and the solution was added with 2-mercaptoethanol (390 mg, 5.0
mmol) and 4-methylbenzenesulfonic acid (33 mg, 0.20 mmol). The reaction was
carried out in the Dean-Stark reactor at refluxing temperature to remove water for 3
hours. After cooling to room temperature, the mixture was added with diethyl ether
(30 mL) and washed with saturated sodium bicarbonate solution (20 mL) and brine
(20 mL) three times respectively. The organic layer was dried with anhydrous Na2SO4,
followed by evaporation of the solvent to obtain crude 4-(1,3-oxathiolan-2-yl)
benzaldehyde (3) as a yellow oil in 89% yield. 1H-NMR (600 MHz, CDCl3), δ (ppm):
10.02 (s, 1H), 7.88 (d, J = 7.8 Hz, 2H), 7.61 (d, J = 8.4 Hz, 2H), 6.12 (s, 1H),
4.57-4.54 (m, 1H), 4.04-4.00 (m, 1H), 3.30-3.21 (m, 2H). 13C-NMR (150 MHz,
CDCl3), δ (ppm): 191.76, 146.35, 136.38, 129.91. 126.92, 86.01, 72.29, 34.06. HRMS
+
(ESI): m/z calcd for C10H11O2S1 [M+H]+ 195.0474, found: 195.0475. IR (KBr, cm-1):
3383(w), 2872(m), 1697(s), 1608(s), 1387(m), 1302(m), 1203(s), 1066(s), 1016(m),
977(m), 806(s), 739(m), 480(w).
Sodium hydride (163 mg, 6.8 mmol) and 2 (296 mg, 0.68 mmol) were dissolved in
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tetrahydrofuran (30 mL) in a two-necked flask, and the solution contained in
two-necked flask was fulfilled with nitrogen atmosphere and cooled to 0 °C. Then the
solution of 3 (269 mg, 1.4 mmol) in tetrahydrofuran (10 mL) was added to the reactor
and the solution was stirred under 0 °C for 12 h. Crude product can be purified by
extraction with dichloromethane and recrystallization with hexane/chloroform to
obtain OPV-MEP as dark yellow powder in 68% yield. MP: 230.6-232.3 °C.
1H-NMR (600 MHz, CDCl3), δ (ppm): 7.55-7.45 (m, 10H), 7.13-7.10 (m, 4H), 6.07 (s,
2H), 4.57-4.54 (m, 2H), 3.99-3.94 (m, 2H), 3.93 (s, 6H), 3.32-3.28 (m, 2H), 3.23-3.20
(m, 2H). 13C-NMR (150 MHz, CDCl3), δ (ppm): 151.53, 138.25, 128.45, 126.95,
126.60, 123.65, 109.17, 86.90, 71.92, 56.35, 34.04. HRMS (ESI): m/z calcd for
+
C30H31O4S2 [M+H]+ 519.1658, found: 519.1665. IR (KBr, cm-1): 3437(w), 2933(w),
2858(w), 1603(w), 1495(m), 1460(m), 1408(m), 1340(w), 1209(s), 1040(s), 968(m),
854(m), 812(w), 745(w), 692(w), 536(w).
2.3. Two-photon fluorescence measurement
Two-photon absorption cross-section was measured by a two-photon excited
fluorescence method using rhodamine B as the reference. The femtosecond laser pulse
from a Ti: sapphire system (690-850 nm, Tsunami) was used as the light source. All
measurements were carried out in air at room temperature. Two-photon absorption
cross-section was calculated by the following equation:
Φref cref nref
F
δ
= δref
Φ
c
n F
ref
here the subscripts ref stands for the reference molecule. δ is the value of
two-photon absorption cross-section; c is the solution concentration; n is the refractive
index of the solution; F is the two-photon fluorescence integral intensities of the
solution emitted at the exciting wavelength; and Φ is the fluorescence quantum yield.
The δref value of the reference is taken from the literature [31].
2.4. Cell cytotoxicity and imaging
To
test
the
cytotoxicity
of
the
probe
OPV-MEP,
MTT
(5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) assay was performed [32].
BV-2 cells were plated in 96-well flat-bottomed plates and incubated for 24 h at 37 °C
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under 5% CO2 prior to the OPV-MEP treatment. Then DMEM (Dulbecco’s Modified
Eagle Medium) with 10% FBS (Fetal Bovine Serum) was removed and replaced with
fresh DMEM, and aliquots of OPV-MEP stock solutions (1 mM DMSO) were added
to obtain final concentrations of 0.1, 1 and 10 µM, respectively. The treated cells were
incubated for 24 h at 37 °C under 5% CO2. Subsequently, cells were treated with 5
mg/mL MTT (40 µL/well) and incubated for an additional 4 h (37 °C, 5% CO2). Then
the cells were dissolved in the mixture of DMSO (150 µL/well), and the absorbance at
570 nm was recorded. The cell viability (%) was calculated according to the following
equation:
Cell Viability=OD570(Sample)/OD570(Control)
where OD570 (Sample) represents the optical density of the wells treated with
various concentration of OPV-MEP and OD570 (Control) represents that of the wells
treated with DMEM plus 10% FBS. The percentage of cell survival values was
relative to untreated control cells.
For two-photon bio-imaging, BV-2 cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS)
(penicillin/streptomycin 100 µg/mL) at 37 °C in a humidified atmosphere with 5%
CO2 and 95% air. Cytoxicity assays showed that the OPV-MEP was safe enough for
two-photon bio-imaging at low concentrations, so that the cells were incubated with 1
µM OPV-MEP at 37 °C under 5% CO2 for 30 min. The cells were washed once and
bathed in DMEM containing no FBS prior to imaging and/or HOCl addition. Then
HOCl was added in the growth medium for 10 min at 37 °C, and the cells were
washed three times with PBS buffer.
For the detection of endogenous HOCl, microglia BV-2 cells were cultured with 10%
fetal bovine serum (FBS) (penicillin/streptomycin 100 µg/mL) exposed with 100
ng/mL LPS at 37 °C in a humidified atmosphere with 5% CO2 and 95% air. Then the
cells were incubated with 1 µM OPV-MEP at 37 °C under 5% CO2 for 30 min, and
washed 3 times with PBS buffer.
Two-photon fluorescence microscopy images of labelled cells were obtained on a
FV1000MPE confocal microscope (Olympus) by exciting the probe with a
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mode-locked titanium-sapphire laser source set at 740 nm.
3. Results and Discussion
3.1. Design and synthesis of probe OPV-MEP
Scheme 1. Synthesis route and proposed sensing mechanism of OPV-MEP. Reagents and
conditions: (a) HCHO, HCl, 1,4-dioxane, H2O, r.t., 4 h; (b) triethyl phosphite, 80 °C, 2 h; (c)
2-mercaptoethanol, p-toluenesulfonic acid, benzene, reflux, 3 h; (d) NaH, THF, 0 °C, 12 h.
For two-photon materials, two-photon action cross-section is the key factor
representing the two-photon fluorescence brightness [33]. Thus it was desired to
design some small two-photon fluorescent probes with large two-photon action
cross-section for HClO detection in biological systems. Toward this goal, we have
designed a new small fluorescent probe with Oligo (p-phenylene vinylene) (OPVs) as
the fluorescent skeleton. OPVs derivative are reported with a large two-photon active
absorption cross-section and high fluorescence quantum yield and as well as good
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photo-stability [34, 35]. Our previous research has suggested that the introduction of
electron-donating and electron-withdrawing moieties into the OPVs molecule could
significantly increase the two-photon absorption probability [36]. Based on this
understanding, we designed aldehyde terminated OPV as TPA fluorescence dye.
Protection of the two aldehyde groups using 2-mercaptoethanol would break D-π-A
structure and produce the oxathiolane group which could be deprotected by HOCl to
recover the aldehyde groups. We therefore used this mechanism to realize HOCl
sensing.
The probe OPV-MEP was synthesized through four simple steps (Scheme 1) and
1
characterized by H NMR, 13C NMR and HRMS (Fig. S1-S3). OPV-MEP was
effectively prepared from reaction of precursor 2 and 3 with sodium hydride. The
compound 3 was synthesized through the protection of commercially available
compound 1,4-phthalaldehyde. The compound 2 was readily synthesized by
1,4-dimethoxybenzene and subsequent reaction of intermediate 1 with triethyl
phosphite.
The proposed detection mechanism of probe OPV-MEP to HOCl was outlined in
the Scheme 1, OPV-MEP is initially oxidized into intermediate sulfoxide compound
4 and sulphone compound 5 by HOCl, and finally hydrolyzed to generate OPV-CHO
with cleavage of the oxathiolane rings. The probe can effectively respond to HOCl
rather than other ROS, in good agreement with previous reported probes with
thioether as recognition moieties [23,37-38]. Mass spectra was used to follow the
deprotecion process of the oxathiolane group by HClO. A peak corresponding to
[OPV-CHO + H]+ appeared at m/z = 399.1599 after adding of HClO to the solution
of OPV-MEP, which demonstrating the conversion of each of the oxathiolane groups
into aldehyde moieties (Fig. S4).
3.2. Absorption and fluorescence properties of OPV-MEP for sensing HOCl
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Fig. 1. UV-Vis titration spectra of OPV-MEP (10 µM) upon addition of HOCl (0-50 µM) in
PBS/EtOH solution (PBS/EtOH = 1/1, v/v, pH 7.4, containing 0.25% THF). Inset: photographs of
OPV-MEP solution before (left) and after (right) addition of HOCl in visible light.
The UV-Vis and one-photon fluorescent spectra of OPV-MEP were determined in
PBS/EtOH solution buffered to physiological pH (0.1 M PBS, PBS/EtOH = 1/1, v/v,
pH 7.4, containing 0.25% THF). The photophysical properties of the OPV-MEP and
OPV-CHO were investigated and summarized in Table S1. As shown in Fig. 1, the
UV-Vis spectrum of OPV-MEP exhibited the maximum absorption band centered at
400 nm (ε = 4.59 × 104 cm−1 mol−1). Upon the addition of HOCl into the solution, the
intensity of the absorption band at 400 nm decreased gradually, and a new absorption
band centered at 420 nm occurred which was identical to the absorption spectrum of
the supposed product OPV-CHO (Fig. S5). This red-shift (about 20 nm) of the
maximum absorption band resulted from the expansion of the π-conjugate system,
which could also be an evidence for the formation of aldehyde group. In addition, the
color of OPV-MEP solution in visible light (shown in the inset of Fig. 1) turned from
green to yellow after the addition of HOCl. The result displayed that the probe could
be a colorimetric probe monitoring HOCl by direct visualisation.
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Fig. 2. Fluourescence titration spectra (a) and corresponding fluorescence intensity ratio (b) of 0.5
µM OPV-MEP upon the addition of HOCl (0- 2.5 µM) in PBS/EtOH solution (PBS/EtOH = 1/1,
v/v, pH 7.4, containing 0.25% THF, λex= 410 nm). Inset : (a) Change in the fluorescence of
OPV-MEP before (left) and after (right) the addition of 50 µM HOCl, (b) the fluorescence
intensity ratio by adding HOCl (0- 160 nM).
The fluorescence spectrum of OPV-MEP exhibited the maximum emission band
centered at 446 nm under 410 nm excitation (Fig. 2). In order to evaluate the
quantitative detection capacity of OPV-MEP, fluorescence titration experiments were
carried out with the addition of HOCl (0-2.5 µM) into 0.5 µM probe in EtOH-PBS
buffer solution (0.1 M PBS, PBS/EtOH = 1/1, v/v, pH 7.4, containing 0.25% THF).
As shown in Fig. 2a, with the increasing amount of HOCl added into the solution
from 0-2.5 µM, the fluorescence intensity at 446 nm decreased, and a new emission
band centered at 534 nm appeared. Notably, there was a good linear relationship
between the fluorescence intensity ratio (I534nm/I446nm) and the concentration of HOCl.
Besides, the solution of OPV-MEP could be observed with a distinguishable
fluorescence change from blue to yellow by direct visualization under 365 nm
irradiation after the addition of HOCl. The result shows that OPV-MEP could make
quantitatively responses towards HOCl at nanomolar level with high sensitivity.
Furthermore, the response time of the probe to HClO was also investigated (Fig. S6),
in which the OPV-MEP displayed fast and complete response towards HClO within
30 seconds.
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Fig. 3. The fluorescence spectra of OPV-MEP before and after the addition of HOCl in
PBS/EtOH solution (pH 4 to 10).
In order to explore the detection effect of the probe in physiological pH, we also
studied its response to HOCl in PBS/EtOH solution with different pH values ranging
from 4 to 10. As shown in Fig .3, the fluorescence intensity of OPV-MEP kept a
constant value at pH 4 to 10. After the addition of HOCl, the emission ratio (F534nm
/
F446nm) became remarkably higher. These results demonstrate that the probe can
response to HOCl over a broad pH range.
3.3. The selectivity
To investigate the selectivity of OPV-MEP to HOCl under physiological
conditions, the fluorescence response of the probe was investigated with the addition
of other competing ROS and reactive nitrogen species (RNS) in the same conditions.
To a 0.5 µM solution of OPV-MEP, typical ROS and RNS including ·OH, 1O2, H2O2,
ONOO- , NO, TBO· and TBHP were added respectively and the fluorescence spectra
were recorded after 30 minutes. As shown in Fig.4, the addition of HOCl to
OPV-MEP resulted in a remarkable change in fluorescence spectrum. However,
OPV-MEP exhibited no response to other ROS and RNS, and even H2O2 as the
precursor of HOCl also did not induce any observable fluorescence variation of the
probe. Thus, OPV-MEP is confirmed to be a highly selective probe for HOCl
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detection in biological system.
Fig. 4. Fluorescence ratio of probe OPV-MEP (0.5 µM) in response to HOCl (2.5 µM) and
various ROS/RNS (2.5 µM) after 30 min in pH 7.4 PBS/EtOH (1:1, v/v, λex= 400 nm, containing
0.25% THF).
3.4. Two-photon absorption properties
Most of the previously reported two-photon probes suffered with modest or small
two-photon absorption action cross-section (Φδ usually less than 1000 GM), which
greatly limit their practical application. Therefore, it was valuable to develop an
efficient two-photon probe for monitoring HOCl with a large turn-on signal. In order
to verify the capability of OPV-MEP as a two-photon probe, the two-photon
absorption properties of OPV-MEP before and after the addition of HOCl were
determined using two-photon excitation fluorescence method with rhodamine B as the
reference. Two-photon absorption cross-section (δ) of OPV-MEP and OPV-CHO
were determined in the wavelength range 700-800 nm in THF. As shown in Fig.5,
over the measured range, the maximum TP action cross-section (δmax) value of
OPV-MEP is calculated to be 105.2 GM (1 GM= 10-50 cm4 s·photon-1) at 740 nm,
while the δmax value of the reaction product OPV-CHO is 2020.5 GM at 740 nm,
which contributed to the electronic structure of OPV skeleton changed from “D–π–D–
π–D” to “A–π–D–π–A”. According to the fluorescence quantum yield of OPV-MEP
and OPV-CHO were 0.75 and 0.56, the two-photon action cross-section of
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OPV-MEP and OPV-CHO were calculated to be 78.9 GM and 1131.5 GM
respectively. As a result, the two-photon action cross-section of the probe enhanced
nearly 15-fold upon the addition of HOCl, which showed that OPV-MEP could serve
as a two-photon “turn-on” probe for monitoring HOCl.
Fig. 5. Two-photon absorption action cross-section of OPV-MEP and reaction product
OPV-CHO in THF solution
3.5. Cell imaging
Encouraged with the fast and highly selective response of the probe to HOCl, we
then sought to evaluate its potential biological application in bio-imaging. To
demonstrate the capability of OPV-MEP for the detection of HOCl in live cells upon
two-photon excitation, murine BV-2 microglial cells were incubated with 1 µM
OPV-MEP for 30 min at 37 °C in PBS buffer. As shown in Fig. 6(a-c), there were
negligible fluorescence signal observed in the green emission channel of 490-540 nm
for OPV-MEP under the excitation at 740 nm. After the addition of HOCl and
incubation for another 10 minutes, intense fluorescence signals were emerged at the
cytoplasm area of BV-2 cells, which indicated that OPV-MEP could be an effective
tool for two-photon imaging of exogenous HOCl with good permeability in living
cells. In addition, the bright-field images demonstrated that BV-2 cells were highly
viable after treating with OPV-MEP during the imaging experiments, indicating
relative low cytotoxicity of the probe towards living cells.
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Fig. 6. Two-photon image of BV-2 cells incubated with OPV-MEP before (a-c) and after (d-f)
addition of HOCl (λex = 740 nm, green channel: 490- 540 nm). (a and d) Bright-Field images of
the cells. (b and e) Fluorescence images of the cells. (c) The overlap of (a) and (b). (f) The overlap
of (d) and (e).
Fig. 7. Two-photon image of OPV-MEP in BV-2 cells incubated without (a-c) and with (d-f) the
stimulation of LPS (λex= 740 nm, green channel: 490- 540 nm). (a and d) Bright-Field images of
the cells. (b and e) Fluorescence images of the cells. (c) The overlap of (a) and (b). (f) The overlap
of (d) and (e).
To investigate the capability of OPV-MEP for the detection of endogenous HOCl,
lipopolysaccharide (LPS) was employed as the stimulation reagent for generating
HOCl in murine BV-2 microglial cells. The experiments were performed by
incubating LPS stimulated cells with OPV-MEP for 30 min at 37 °C in PBS buffer.
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Then the images of the cells were taken using TPM and clear fluorescence signal of
the cells were observed at the optical window of 490-540 nm under 740 nm light
excitation (Fig.7 d-f). In contrast, cells without the pre-treatment of LPS exhibited
weak fluorescence signal as shown in Fig.7 a-c. Furthermore, two-photon
fluorescence spectroscopy of LPS stimulated BV-2 cells were also investigated. After
treatment of LPS for 48 h, OPV-MEP was added to the culture medium and the cells
were incubated for another 30 min at 37 °C. As shown in Fig.7 d-f, two-photon
fluorescence signal intensity of the probe in cytoplasm increased significantly after
stimulated by LPS, compared to the one without LPS stimulation. Besides, it was
clearly shown that a numeric enhancement of BV-2 cells was observed after LPS
treatment, and no significant decrease in cell viability was observed.
3.6. Cell cytotoxicity
To demonstrate the practical use of OPV-MEP as a HOCl sensor for bio-imaging
in living cells, the cytotoxicity of OPV-MEP was evaluated. MTT assay was
conducted with different probe concentration ranging from 0.1 to 10 µM and BV-2
mouse microglia cell line was chosen for incubation. As shown in Fig. 8, more than
90% survival rate was observed, which showed great cell viability of OPV-MEP. The
result indicated that OPV-MEP exhibited low cytotoxicity to BV-2 cells for 24 h
incubation period. Therefore, the desired low cytotoxicity of OPV-MEP could render
it safe to use for two-photon HOCl imaging in living cells.
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Fig. 8. Cell viability of BV-2 under different concentration of OPV-MEP for 24h.
4. Conclusions
In summary, we developed a new small molecule fluorescent probe OPV-MEP for
monitoring hypochlorous acid based on the oligo (p-phenylene vinylene) two-photon
platform. The addition of hypochlorous acid to OPV-MEP results in a large turn-on
fluorescence signal in two-photon experiment. Furthermore, the probe displayed fast
response, high selectivity for HOCl over other ROS as well as pH independence. Cell
imaging experiments also confirmed the probe with good cell penetrability and low
cytotoxicity. Benefited by these desired sensing properties and biocompatibility, the
probe was successfully applied to the determination of exogenous and endogenous
hypochlorous acid in living BV-2 cells for two-photon fluorescence imaging. We
believe that OPV-MEP provides a promising tool to the further investigation of HOCl
in biological research and clinical diagnoses.
Acknowledgments
The authors acknowledge the support from National Natural Science Foundation of
China (NSFC. 51525303, 21602093, 31600831, 21572086), the Fundamental
Research Funds for the Central Universities and 111 Project.
References
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ACCEPTED MANUSCRIPT
[1] Zhang JJ, Zhen X, Upputuri PK, Pramanik M, Chen P, Pu KY. Activatable
photoacoustic nanoprobes for In vivo ratiometric imaging of peroxynitrite. Adv
Mater 2017;29(6):1604764.
[2] Zhen X, Zhang CW, Xie C, Miao QQ, Lim KL, Pu KY. Intraparticle energy level
alignment of semiconducting polymer nanoparticles to amplify
chemiluminescence for ultrasensitive in vivo imaging of reactive oxygen species.
ACS Nano 2016;10(6):6400-6409.
[3] Pu KY, Shuhendler AJ, Jokerst JV, Mei JG, Gambhir SS, Bao ZN, Rao JH.
Semiconducting polymer nanoparticles as photoacoustic molecular imaging
probes in living mice. Nature Nanotech 2014;9(3):233-239.
[4] Bauer G. Reactive oxygen and nitrogen species: efficient, selective, and
interactive signals during intercellular induction of apoptosis. Anticancer Res
2000;20(6B):4115-4139.
[5] Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing.
Nature 2000;408(6809):239-247.
[6] Chen XQ, Tian XZ, Shin I, Yoon J. Fluorescent and luminescent probes for
detection of reactive oxygen and nitrogen species. Chem Soc Rev 2011;40(9):
4783-4804.
[7] Chen XQ, Wang F, Hyun JY, Wei TW, Qiang J, Ren XT, Shin I, Yoon J. Recent
progress in the development of fluorescent, luminescent and colorimetric probes
for detection of reactive oxygen and nitrogen species. Chem Soc Rev
2016;45(10):2976-3016.
[8] Lapenna D, Cuccurullo F. Hypochlorous acid and its pharmacological antagonism:
An update picture. Gen Pharmacol 1996;27(7):1145−1147.
[9] Marcinkiewicz J,Chain B, Nowak B, Grabowska A, Bryniarski K, Baran J.
Antimicrobial and cytotoxic activity of hypochlorous acid: interactions with
taurine and nitrite. Inflamm Res 2000;49(6):280-289.
[10]Klebanoff SJ. Myeloperoxidase: friend and foe.
2005;77(5):598-625.
J
Leukoc Biol
[11]Pullar JM, Vissers MCM, Winterbourn CC. Living with a killer: the effects of
17

ACCEPTED MANUSCRIPT
hypochlorous acid on mammalian cells. IUBMB Life 2000;51(4-5):259-266.
[12]Andersen JK. Oxidative stress in neurodegeneration: cause or consequence? Nat
Med 2004;10(7):S18-S25.
[13]Yap YW, Whiteman M, Cheung NS. Chlorinative stress: An under appreciated
mediator of neurodegeneration? Cell Signalling 2007;19(2):219-228.
[14]Jeitner TM, Kalogiannis M, Krasnikov BF, Gomlin I, Peltier MR, Moran GR.
Linking Inflammation and Parkinson Disease: Hypochlorous Acid Generates
Parkinsonian Poisons. Toxicol Sci 2016;151(2):388-402.
[15]Liang LJ, Liu C, Jiao XJ, Zhao LC, Zeng XS. A highly selective and sensitive
photoinduced electron transfer (PET) based HOCl fluorescent probe in water and
its endogenous imaging in living cells. Chem Commun 2016;52(51):7982-7985.
[16]Zhou J, Li LH, Shi W, Gao XH, Li XH, Ma HM. HOCl can appear in the
mitochondria of macrophages during bacterial infection as revealed by a sensitive
mitochondrial-targeting fluorescent probe. Chem Sci 2015;6(8):4884-4888.
[17]Zhang YR, Meng N, Miao JY, Zhao BX. A Ratiometric Fluorescent probe based
on a through-bond energy transfer (TBET) system for imaging HOCl in living
cells. Chem Eur J 2015;21(52):19058-19063.
[18]Ding SS, Zhang Q, Xue SH, Feng GQ. Real-time detection of hypochlorite in tap
water and biological samples by a colorimetric, ratiometric and near-infrared
fluorescent turn-on probe. Analyst 2015;140(13):4687-4693.
[19]Cheng GH, Fan JL, Sun W, Cao JF, Hu C, Peng XJ. A near-infrared fluorescent
probe for selective detection of HClO based on Se-sensitized aggregation of
heptamethine cyanine dye. Chem Commun 2014;50(8):1018-1020.
[20]Li GP, Zhu DJ, Liu Q, Xue L, Jiang H. A strategy for highly selective detection
and imaging of hypochlorite using selenoxide elimination. Org Lett
2013;15(8):2002-2005.
[21]Wang BH, Chen D, Kambam S, Wang F, Wang Y, Zhang W, Yin J, Chen HY,
Chen XQ. A highly specific fluorescent probe for hypochlorite based on
fluorescein derivative and its endogenous imaging in living cells. Dyes Pigments
2015;120:22-29.
18

ACCEPTED MANUSCRIPT
[22]Ma H, Song B, Wang YX, Liu CL, Wang X, Yuan JL. Development of
organelle-targetable europium complex probes for time-gated luminescence
imaging of hypochlorous acid in live cells and animals. Dyes Pigments
2017;140:407-416.
[23]Yuan L, Wang L, Agrawalla BK, Park SJ, Zhu H, Sivaraman B, Peng JJ, Xu QH,
Chang YT. Development of targetable two-photon fluorescent probes to image
hypochlorous acid in mitochondria and lysosome in live cell and inflamed mouse
model. J Am Chem Soc 2015;137(18):5930-5938.
[24]Gao YT, Feng GX, Jiang T, Goh CC, Ng LG, Liu B, Li B, Yang L, Hua JL, Tian
H. Biocompatible nanoparticles based on Diketo-Pyrrolo-Pyrrole (DPP) with
aggregation-induced red/NIR emission for in vivo two-photon fluorescence
imaging. Adv Funct Mater 2015;25(19):2857-2866.
[25]Kim D, Ryu HG, Ahn KH. Recent development of two-photon fluorescent probes
for bioimaging. Org Biomol Chem 2014;12(26):4550-4566.
[26]Li L, Zhang CW, Chen GYJ, Zhu BW, Chai C, Xu QH, et al. A sensitive
two-photon probe to selectively detect monoamine oxidase B activity in
Parkinson's disease models. Nat Commun 2014;5:3276-85.
[27]Zhou L, Zhang X,Wang Q, Lv Y, Mao G, Luo A, et al. Molecular engineering of a
TBET-based two-photon fluorescent probe for ratiometric imaging of living cells
and tissues. J Am Chem Soc 2014;136:9838-9841.
[28]Kim HM, Cho BR. Two-photon materials with large two-photon cross sections.
Structure-property relationship. Chem Commun 2009;2:153-164.
[29]H.M. Kim, B.R. Cho, Small-molecule two-photon probes for bioimaging
applications, Chem. Rev. 115 (2015) 5014-5055.
[30]Wu GF, Zeng F, Wu SZ. A water-soluble and specific BODIPY-based fluorescent
probe for hypochlorite detection and cell imaging. Anal Methods 2013;5(20):
5589-5596.
[31]Xu C, Webb WW. Measurement of two-photon excitation cross sections of
molecular fluorophores with data from 690 to 1050 nm. J Opt Soc Am B
1996;13(3):481-491.
19

ACCEPTED MANUSCRIPT
[32]Cheng D, Pan Y, Wang L, Zeng ZB, Yuan L, Zhang XB, Chang YT. Selective
visualization of the endogenous peroxynitrite in an inflamed mouse model by a
mitochondria-targetable two-photon ratiometric fluorescent probe. J Am Chem
Soc 2017;139(1):285-292.
[33]Pawlicki M, Collins HA, Denning RG, Anderson HL. Two-Photon absorption and
the design of two-photon dyes. Angew Chem Int Ed 2009;48(18):3244-3266.
[34]Gao F, Liao Q, Xu ZZ, Yue YH, Wang Q, Zhang HL, Fu HB. Strong two-photon
excited fluorescence and stimulated emission from an organic single crystal of an
Oligo(Phenylene Vinylene). Angew Chem Int Ed 2010;49(4):732-735.
[35]Pond SJK, Tsutsumi O, Rumi M, Kwon O, Zojer E, Bredas JL, Marder SR, Perry
JW. Metal-ion sensing fluorophores with large two-photon absorption cross
sections: aza-crown ether substituted donor-acceptor-donor distyryl benzenes. J
Am Chem Soc 2004;126(30):9291-9306.
[36]Sun CL, Li J, Geng HW, Li H, Ai Y, Wang Q, Pan SL, Zhang HL. Understanding
the unconventional effects of halogenation on the luminescent properties of
Oligo(Phenylene Vinylene) molecules. Chem Asian J 2013;8(12):3091-3100.
[37]Xu Q, Lee KA, Lee S, Lee KM, Lee WJ, Yoon J. A highly specific fluorescent
probe for hypochlorous acid and its application in imaging microbe-induced
HOCl production. J Am Chem Soc 2013;135(26):9944-9949.
[38]Hawkins CL, Pattison DI, Davies MJ. Hypochlorite-induced oxidation of amino
acids, peptides and proteins. Amino Acids 2003;25(3):259-274.
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A Highly Selective Two-Photon Probe with Large Turn-On
Signal for Imaging Endogenous HOCl in Living Cells
a
a
b
b
a
Jingjing Cao , Dong-Ming Jiang , Xin Ren , Ting Li , Xiao-Ting Gong ,
Yong-Rui Yang a, Zhu-Guo Xu a, Chun-Lin Sun a, Zi-Fa Shi a, Shengxiang Zhang
b, * and Hao-Li Zhang a, *
a State Key Laboratory of Applied Organic Chemistry (SKLAOC); Key Laboratory of
Special Function Materials and Structure Design (MOE); College of Chemistry and
Chemical Engineering, Lanzhou University, Lanzhou, 730000, P. R. China
b School of Life Sciences, Lanzhou University, Lanzhou 730000, P. R. China
Tel: +86-0931-8912365 (H.Z.)
E-mail: haoli.zhang@lzu.edu.cn (H.Z.), sxzhang@lzu.edu.cn (S.Z.)
Highlights
ó
ó
ó
Large two-photon action cross-section of 1131.5 GM with small molecular
weight.
High specificity, fast response for HOCl fluorescence monitoring in a broad pH
range.
Selective imaging of endogenous HOCl in microglia BV-2 cells with low
cytotoxicity.