An NBD–NH2 fluorescent probe for bioimaging: existence of a specific detection of ClO?

Article abstract of DOI:10.1007/s00706-018-2143-9

Abstract: A fluorescent probe 4-amino-7-nitrobenz-2-oxa-1,3-diazole NBD–NH₂ for critical sensing hypochlorous (ClO^?) has been developed in this paper. Probe NBD–NH₂ exhibited excellent sensitivity and high selectivity toward detection of ClO^? in CH₃CH₂OH/HEPES solution (1:4, v/v, 10?mM, pH 7.4), where the detection limit as low as 40?nM. The green fluorescence of NBD–NH₂ was distinctly quenched by addition of ClO^?. In addition, the new probe was also applied for bioimaging using HeLa cells and exist a critical detection of ClO^? in living cells. Graphical abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00706-018-2143-9

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https://doi.org/10.1007/s00706-018-2143-9(0123456789(). -, vo Vl
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ORIGINAL PAPER
An NBD–NH2 fluorescent probe for bioimaging: existence of a specific
detection of ClO2
Yunxia Yang1 Di Zhang2 Mengsheng Xu1 Jianhong Wang1 Jipeng Chen1 Le Wang1
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Received: 25 October 2017 / Accepted: 1 January 2018
Ó Springer-Verlag GmbH Austria, part of Springer Nature 2018
Abstract
A fluorescent probe 4-amino-7-nitrobenz-2-oxa-1,3-diazole NBD–NH2 for critical sensing hypochlorous (ClO-) has been
developed in this paper. Probe NBD–NH2 exhibited excellent sensitivity and high selectivity toward detection of ClO- in
CH3CH2OH/HEPES solution (1:4, v/v, 10 mM, pH 7.4), where the detection limit as low as 40 nM. The green fluorescence
of NBD–NH2 was distinctly quenched by addition of ClO-. In addition, the new probe was also applied for bioimaging
using HeLa cells and exist a critical detection of ClO- in living cells.
Graphical abstract
NH2
NO
Cl
N
N
N
N
N
N
NH3'H2O
CH3OH
ClO-
O
O
O
NO2
NO2
NO2
NBD-NH2
NBD-NO
NBD-Cl
Keywords NBD–NH2 Á Fluorescent probe Á Hypochlorous
Introduction
in living organisms [3–5]. However, excessive or mis-
placed generation of HClO always can produce many
human diseases or tissue damage. For example, neuron
degeneration can cause atherosclerosis [6–9]. Therefore, it
is very important to develop different efficient protocols to
detect the HClO/ClO-.
Hypochlorous acid (HClO)/hypochlorite (ClO-), as one of
important reactive oxygen species (ROS), play an indis-
pensable role in varieties of physical and biological sys-
tems [1, 2]. Endogenous ClO- usually generated from
H2O2 and Cl- by the catalysis of myeloperoxidase (MPO)
Based on this, numbers of techniques have been studied
to detect HClO/ClO-, such as electroanalysis, potentiom-
etry, spectrophotometry, and chemiluminescent [10–13].
Among various developed effective methods, the fluores-
cence probe method is a well-known method because of its
simplicity, sensitivity, and potential for biological imaging
features [14]. Recently, many small molecular fluorescent
probes for HClO/ClO- sensing have been studied [15–24].
However, there are still some challenges to optimize these
probes, such as poor stability for oxidants [25], difficult to
prepare [26], which limit their applications. It is necessary
to develop different probes to detection of HClO/ClO-. As
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00706-018-2143-9) contains supplementary
material, which is available to authorized users.
& Le Wang
Wangle316@sues.edu.cn
1
College of Chemistry and Chemical Engineering, Shanghai
University of Engineering Science, Shanghai 201620, China
2
Henan Cereal Quality and Safety Testing Key Laboratory,
Institute of Quality Standards and Testing Technology for
Agro-Products, Henan Academy of Agricultural Sciences,
Zhengzhou 450002, China
we
know,
7-nitrobenz-2-oxa-1,3-diazole
(NBD)
123

Y. Yang et al.
fluorophore has been widely studied due to its long emis-
sion wavelength, nice cell permeability, and easy to mod-
ification [27]. Herein, we designed the new fluorescent
probe NBD–NH2 to detect HClO/ClO-. As shown in
Scheme 1, amino group was introduced to 4-chloro-7-ni-
trobenz-2-oxa-1,3-diazole NBD–Cl system to construct a
new probe NBD–NH2 for critical detection of HClO/ClO-
based on the proposed mechanism of amido-oxidized
nitroso-group by hypochlorites. As expected, the simple
modified probe NBD–NH2 exhibited a critical detection of
HClO/ClO- in CH3CH2OH/HEPES (v/v, 1:4, 10 mM, pH
7.4).
Results and discussion
Synthesis and characterization
Fig. 1 UV–Vis absorption of probe NBD–NH2 (10 lM) with NaClO
(100 lM) and other various analytes (100 lM) in CH3CH2OH/
HEPES (1:4, v/v, 10 mM, pH 7.4)
As shown in Scheme 1, probe NBD–NH2 was prepared
according to the reported procedures [28, 29], and the
structure of probe NBD–NH2 was well characterized by 1H
NMR, 13C NMR, and HR-MS (S1–S3).
originally characteristic peak at 468 nm. A remarkable
reduction of UV–Vis absorption can be observed when
NaClO (10 equiv.) was added in this system. However,
other biologically relevant analytes almost could not result
in any evident change under the same testing conditions.
The stock solution of probe NBD–NH2 (1 mM,
CH3CH2OH) was firstly prepared, and then was diluted to
relevant concentration for further study. The fluorescence
and absorbance studies were carried out in aqueous solu-
tion to verify the selectivity of NBD–NH2 to ClO-. All the
experiments described here were studied under the condi-
tion of CH3CH2OH/HEPES (1:4, v/v, 10 mM, pH 7.4),
which can provide the best results [30].
Fluorescence spectral responses of NBD–NH2
to ClO2
Selectivity
As shown in Fig. 2, we analyzed the fluorescence probe
NBD–NH2 selectivity of hypochlorous acid among ROSs
and other anions. The maximum fluorescence response of
free probe NBD–NH2 was about at 549 nm. A remarkable
Absorbance spectra responses of NBD–NH2
to ClO2
The sensing ability of NBD–NH2 for ClO- was first
investigated by UV–Vis absorption. Upon addition of
NaOCl and other analytes in this reaction system, the
absorption spectra are observed to change and are shown in
Fig. 1, where the free probe NBD–NH2 displayed an
Scheme 1
NH2
Cl
N
N
N
NH3'H
2O
O
O
N
CH3OH
NO2
NO2
NBD-NH2
NBD-Cl
Fig. 2 Fluorescence intensity of NBD–NH2 (549 nm, 10 lM) with
NaClO (100 lM) and other various analytes (10 eq.) in CH3CH2OH/
HEPES (1:4, v/v, 10 mM, pH 7.4) (kex = 465 nm, slit width 5 nm)
123

An NBD–NH2 fluorescent probe…
pH dependence
fluorescence quenching occurred after adding 10 equiv. of
NaClO into in this system. Meanwhile, the green solvent
change into colorless (Fig. 2, inset). Other various bio-
To find a suitable pH span to detect NaClO efficiently, the
fluorescence of NBD–NH2 in various pH values is inves-
tigated and these results are shown in Fig. 4. It was con-
firmed that the free probe NBD–NH2 was pH insensitive at
range of 4–9. After adding NaOCl (10 equiv.), the emis-
sion intensity at 549 nm distinctly quenched within the
pale of pH 6.0–8.0. Just as shown in Fig. 4, both NBD–
NH2 and NBD–NH2 ? NaClO were pH insensitive in a
relatively broad pH of biologically relevant, which indi-
cating that the probe is compatible with adhibitioning
under physiological conditions.
logically relevant analytes (such as F-, Cl-, Br-, I-, S2-
,
,
3-
SO42-, HSO4-, NO3-, NO2-, CO32-, HCO3-, PO4
H2PO4-, HPO42-, H2O2, O2-, TBHP, Cys, GSH, 1O2, NO,
and ÁOH) have no evident fluorescence change under the
same testing conditions.
Furthermore, the competition experiments of other
analytes were also conducted to evaluate the reaction
activity of NBD–NH2. As shown in Fig. 3, NBD–NH2 still
resulted in the similar fluorescence quenching when
10 equiv. of NaClO was added into other analytes,
respectively. It is clear that all the tested anions and ROSs/
RNSs have no obviously interference from detecting ClO-
by NBD–NH2. All the above results indicated that probe
NBD–NH2 exists a critical detection property toward
NaClO over other competitive analytes.
Sensitivity
As the detection limit was another important parameter for
a new fluorescent probe, we investigate NBD–NH2 per-
formance in the quantitative detection of NaClO in CH3-
CH2OH/HEPES (1:4, v/v, 10 mM, pH 7.4) and evaluate its
practical applicability. As shown in Fig. 5, the emission
intensity decreases with the increasing of of NaClO
(0–100 lM) at about 549 nm. A good linear fitting was
obtained between fluorescence intensity and NaClO con-
centration (R2 = 0.991), and probe NBD–NH2 showed
lower detection limit that reached to 40 nM [31, 32]. The
result indicated that the probe NBD–NH2 showed a certain
sensitivity toward ClO- that were comparable to the other
fluorescent probes for ClO- (Table S1) [33–38].
Time dependence
Considering reaction efficiency, the time dependence of
probe NBD–NH2 toward NaClO was also investigated. As
shown in Fig. S4, it was obviously that the fluorescence of
the NBD–NH2 with NaClO decreases and then reaches
saturation in about 25 min, while blank solution (only
NBD–NH2) almost has no change under the same test
conditions. As the reaction almost finished in 25 min (see
time-dependence plot), all subsequent experiments were
carried out after 25 min to make the analytes chelate with
the probe sufficiently.
Fig. 4 Fluorescence intensity of free probe NBD–NH2 (549 nm,
10 lM) and in the presence of 10 eq. NaClO in CH3CH2OH/HEPES
(1:4, v/v, 10 mM, pH 7.4) under different pH (10 mM HEPES)
conditions (kex = 465 nm, slit width 5 nm)
Fig. 3 Fluorescence intensity of NBD–NH2 (549 nm, 10 lM) with
NaClO (100 lM) and other various analytes (10 eq.) in CH3CH2OH/
HEPES (1:4, v/v, 10 mM, pH 7.4) (kex = 465 nm, slit width 5 nm)
123

Y. Yang et al.
Bioimaging application
The practical application of probe NBD–NH2 was further
investigated in biological systems [43–45]. Fibered con-
focal fluorescence microscopy (FCFM) imaging using
HeLa cells is carried out and the results are depicted in
Fig. 6. A strong fluorescence phenomenon of probe NBD–
NH2 was observed inside the living HeLa cells (Fig. 6b).
When HeLa cells with NaClO were incubated at 37 °C for
30 min, a remarkable fluorescence quenching can be
observed in Fig. 6d. Furthermore, a bright field transmis-
sion image of HeLa cells treated with NBD–NH2 and
NBD–NH2 ? NaClO also was observed as control, which
demonstrated that the cells viable in the process (Fig. 6a,
c). Hence, the new probe NBD–NH2 has the potential
application for bioimaging in the detection of ClO- in
biological samples.
Fig. 5 Fluorescence emission of probe NBD–NH2 (10 lM) with
various amounts of NaClO in CH3CH2OH/HEPES (pH 7.4). Inset: the
plot of fluorescence intensity of probe NBD–NH2 (549 nm, 10 lM)
with ClO- in CH3CH2OH/HEPES (pH 7.4) (kex = 465 nm, slit width
5 nm)
Conclusion
Proposed sensing mechanism
In summary, a new fluorescent probe NBD–NH2 has suc-
cessfully developed for the critical detection of
hypochlorous (ClO-) over other ROSs/RNSs. Because of
excellent selectivity and sensitivity, low detection limits
(40 nM) detection of hypochlorous (ClO-), probe NBD–
NH2 showed good application prospects, and the prelimi-
nary experimental results demonstrated existence of a
critical detection property of ClO- in biological samples.
Both the UV–Vis and the fluorescence experiments resul-
ted in significant ON–OFF phenomena. According to the
previous reports [39–42], the detection mechanism for
HClO/ClO- probes with amino group was proved that the
amido-oxidized nitroso-group. The form of compound
NBD–NO was also confirmed by ESI–MS analysis
(Fig. S5). When NaClO was added to probe NBD–NH2 in
CH3OH, a new peak 195.06 was appeared, which was
suggested
the
compound
[NBD–NO ? H]?
(calcd = 195.02). Thus, the similar mechanism is prelim-
inarily proposed and is shown in Scheme 2. When the
ClO- was introduced, the free NBD–NO moiety generated
and weak fluorescence emission was released, while probe
NBD–NH2 displayed strong fluorescence emissive at
549 nm.
Experimental
NMR spectra were carried out with a Bruker Avance II
(400 MHz), and chemical shifts are given as ppm. Fluo-
rescence spectra were recorded with the F-7000 FL Spec-
trophotometer (Hitachi, Japan). Absorption spectra were
measured on Agilent Cary 100 spectrometer. HR-MS (ESI)
analyses were carried out with a Thermo HPLC Q-Exactive
with ions given in m/z. HeLa cell fluorescent images were
taken under a LEICA TCS SP8 laser scanning confocal
microscope.
Scheme 2
All solvents and reagents were of analytical grade.
Deionized water was in the experiments throughout. The
solutions of anions were prepared from their sodium salts.
-
Sodium hypochlorite (NaClO), H2O2, and O2 were pre-
pared according to reported literature.
4-Amino-7-nitrobenz-2-oxa-1,3-diazole (NBD–NH2, C6H4N4O3)-
Probe NBD–NH2 was prepared according to the previous
reported procedures with NBD–Cl as starting materials
(Scheme 1). Excess of ammoniumhydroxide (4 cm3, 28% in
water) was added to NBD–Cl (1 mmol) at r.t., drops by
NBD-NH2
NBD-NO
Fluorescence ON
Fluorescence OFF
123

An NBD–NH2 fluorescent probe…
Fig. 6 Confocal fluorescence
images of NaClO with probe
NBD–NH2 in Hela cells. b Hela
cells pretreated NBD–NH2
(10 lM) for 30 min. d Hela
cells were pretreated with probe
NBD–NH2 (10 lM) and then
incubated with NaClO (30 lM)
for 30 min. a, c Bright field
images of b, d, correspondingly
(kex = 465 nm, green channel)
(color figure online)
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drops. The mixture was stirred and kept for 24 h, and then,
the reaction was conducted as usually. The crude product
was further purified by column chromatography (n-hexane/
ethyl acetate, 1/1, Rf = 0.4), providing purified brown red
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