An o-hydroxyl aldehyde structure based naphthalimide derivative: Reversible photochromic properties and its application in ClO? detection in living cells

Article abstract of DOI:10.1016/j.saa.2019.04.049

A bifunctional organic compound 2-butyl-6-hydroxy-1,3-dioxo-2,3-dihydro-1H-benzo[de] isoquinoline-5-carbaldehyde (BHC) with photochromic properties in solid state and probe detection for ClO^? in complete water solution was synthesized and fully characterized. A ‘white–yellow–white’ reversible photochromic behavior could be observed when alternating UV/vis light irradiation on the solid BHC powder. Good fatigue resistance and adjustable bleaching rate were shown when heating conditions changes. In addition, BHC displayed a high selectivity and low detection limit (1.16 × 10^?8 M) for ClO^?. The photoluminescent fluorescence “on-off” recognition result can be easily identified and BHC has been tested for safely imaging living cells and detecting hypochlorite anion in vitro and vivo. A better water solubility of BHC effectively reduces damage caused by organic solvent in cell imaging progress.

Full text of DOI:10.1016/j.saa.2019.04.049

Accepted Manuscript
An o-hydroxyl aldehyde structure based naphthalimide derivative:
Reversible photochromic properties and its application in ClO−
detection in living cells
Xinxue Tang, Hao Sun, Jing Nie, Xiang'en Han, Yun Zhao, Ran
Zhang, Zhonghai Ni
PII:
S1386-1425(19)30430-5
https://doi.org/10.1016/j.saa.2019.04.049
SAA 17057
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 27 February 2019
Revised date:
Accepted
date:
12 April 2019
1
8 April 2019
Please cite this article as: X. Tang, H. Sun, J. Nie, et al., An o-hydroxyl aldehyde structure
based naphthalimide derivative: Reversible photochromic properties and its application in
ClO− detection in living cells, Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy, https://doi.org/10.1016/j.saa.2019.04.049
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ACCEPTED MANUSCRIPT
An o-Hydroxyl Aldehyde Structure Based Naphthalimide Derivative:
−
Reversible Photochromic Properties and Its Application in ClO Detection
in Living Cells
Xinxue Tang, Hao Sun, Jing Nie, Xiang’en Han*, Yun Zhao, Ran Zhang, Zhonghai Ni*
School of Chemical Engineering and Technology, China University of Mining and Technology, Xuzhou221116,
People’s Republic of China;
Corresponding author E-mail: xiangenh@163.com (X. E. Han) and nizhonghai@cumt.edu.cn (Z. H. Ni)
Abstract:
A
bifunctional organic compound 2-butyl-6-hydroxy-1,3-dioxo-2,3-dihydro-1H-benzo[de]
isoquinoline-5-carbaldehyde (BHC) with photochromic properties in solid state and probe detection
−
for ClO in complete water solution was synthesized and fully characterized. A
‘white–yellow–white’ reversible photochromic behavior could be observed when alternating UV/vis
light irradiation on the solid BHC powder. Good fatigue resistance and adjustable bleaching rate
were shown when heating conditions changes. In addition, BHC displayed a high selectivity and
-8
−
low detection limit (1.16 × 10 M) for ClO . The photoluminescent fluorescence "on-off"
recognition result can be easily identified and BHC has been tested for safely imaging living cells
and detecting hypochlorite anion in vitro and vivo. A better water solubility of BHC effectively
reduces damage caused by organic solvent in cell imaging progress.
−
Keywords: Reversible photochromic; Fatigue resistance; ClO detection; Cell imaging; Water
solubility;
1
Introduction
Organic photoactive materials, which could switch their optical signals towards the external
stimulation or output the bright multi-colors in different environments [1], have attracted much
attention for the potential applications in fluorescent sensors, organic light emitting diodes (OLED)
[
2, 3] and biological imaging in the past few decades [4-6]. Among many research fields, exploiting
the inherent diversity of compounds to take full advantages of molecular properties and maximize
the use of materials has been paid much attention by many researchers [7, 8]. Naphthalimide, as a
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typical chromophore, has been extensively designed and widely used as molecular sensor [9] and
cell imaging material [10], which is based on its unique fluorescent properties such as strong
fluorescence, high sensitivity, good light stability and be stable in the physiological pH range [11].
At the same time, the emission signal of such an attractive fluorophore can be detected easily, which
dramatically expand the range of practically useful applications of fluorescent part and make them
potentially used for optical signal storage and collection, like photochromic compounds had done
[
12-14]. These compounds are powerful research objects for bifunctional and multifunctional
material [15].
Since Hirshberg had proposed concept of photochromism, photochromic compounds have been the
focus of high-density information storage and recording functional materials [16, 17]. The use of
photochromic compounds, such as Schiff bases, diarylethene and spiropyran, as photo switching
systems attracts much attention because of their potential ability for application in photonic devices
[
18]. Among various photochromic compounds, Schiff base is one of the most important class of
these materials [19-21]. They have good chemical stability and anti-degradation ability under
illumination, like salicylaldehyde based Schiff base. These compounds may undergo photochromic
behavior in solid state and crystalline state, which expands the application in optical devices and
anti-counterfeiting fields [22]. Their discoloration is a light-induced proton transfer process.
Specifically, it is a proton easy to escape and a pairs of electrons provided by nitrogen atoms in
adjacent positions will attract it. In the excited state, it is relatively prone for proton to transfer from
the oxygen atom in Phenolic hydroxyl to the nitrogen atom, resulting in a color change. In addition
to the nitrogen atom, the oxygen atom and sulfur atom have a similar electron donating structure
[
23, 24]. However, such phenomena is rarely found in an ortho-hydroxy aldehyde compound who
shares the same potential proton transfer ability. In 2017, Chen and his coworkers had designed and
synthesized several naphthaldehyde molecules and tried to find out the excited state intramolecular
proton transfer (ESIPT) between aldehyde and hydroxyl group, moving multiple protons across a
bridging hydrogen bond (H-bond) [25]. Nevertheless, this research was conducted in the polar,
aprotic solvents, which make a big difference compared with the solid state.
Research on the development of photochromic compounds in solid and crystalline states remains
attractive, and this expansion of photochromic compounds based on the ESIPT mechanism will be a
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promising result in more sensors and smart materials application area [26].
In addition to applications of optical devices, many researchers have also focused on bio-probes
these are able to detect various substances, ions and reactive oxygen species (ROS) in cells in order
to efficiently utilize various excellent properties of organic photo luminescent compounds [27-32].
−
Hypochlorite (ClO ), a daily used disinfectant, is an essential member of reactive oxygen species
(ROS) in living organism and plays a vital role in the immune system due to its antibacterial
properties [33-35]. Organisms produce ROS in the mitochondria of phagocytic cells to prevent
-5
-2
inflammatory stimuli, such as host defense against bacterial and fungal infections [36]. 10 -10 M
is usually regarded as a safe hypochlorite concentration range, outside of which may bring about
many health risks like neural degeneration, cardiovascular disease and even cancers [37-42].
Considering real-time and in-situ detection, we speculate that traditional options, potentiometric
−
titration and electrochemical analysis, for detection of ClO are not the best methods since both of
which need complex sample preparation, expensive and complicated instruments and long response
time. That is the main reason we take fluorescent chemo-sensors into consideration for their special
advantages such as simplicity, low cost, high sensitivity, high selectivity, and rapid response time
[
43-47].
−
Fluorescence sensing for real-time visual detection of hypochlorite (ClO ) in vitro and in vivo has
been a hot topic among researchers in recent decades. In 2014, Yin and Yang et al. found a
commercially available fluorescent reagent that be used as hypochlorite probe. Based on the idea of
oxidizing amino groups to imines, they noticed fluorescent emission of probe changed from blue to
green in ethanol when hypochlorite solution added [48]. One year later, Wang et al. designed and
synthesized an ‘off-on’detection probe for the imaging of hypochlorous acid in mitochondria with a
relatively long emission wavelength and detection limit was as low as 17.9 nM in PBS (containing
−
0
.5% DMSO) [49]. In 2017, Huo’s group designed a probe to detect ClO in tap and river water
−
samples by spiking a known amount of standard ClO . Fluorescence quantum yield increased from
0
.0008 to 0.112 when adding hypochlorite to non-fluorescent solution (DMSO: H O, v/v, 1/3) [50].
2
Recently, many scholars have extended the research of molecular probes to the fields of biology and
biological applications [51] and cell imaging materials [52] as well as simultaneous the detection of
endogenous hypochlorous acid [53-55]. In order to accurately tracking HClO formation, efficient
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and effective sensors for real-time monitoring in vivo are imperatively needed. However, most of
the existing probes for HClO detection are poorly soluble in water and require additional organic
solvent when used which is inapplicable in living cells [56-58]. Therefore, it is an urgent demand to
−
unearth new indicators for highly sensitive and accurate detection of ClO in cells and organs.
Herein, we explored a o-hydroxyl aldehyde structure based naphthalimide derivative, named
2
-butyl-6-hydroxy-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-5-carbaldehyde (BHC) that
brought a hydroxyl and an aldehyde to equip naphthalimide core with a proton acceptor and donor
and built a structure that increased the water solubility of organic compound. BHC showed a
−
special selective response to ClO among kinds of common ions. The photoluminescence (PL)
−
intensity and peak wavelengths could be reversibly regulated by UV-vis light, and addition of ClO
solution. Based on these results, we synthesized and characterized BHC that had both photochromic
ability and recognition of hypochlorite in living cells. To the best of our knowledge, this
bifunctional compound was a novel one with a color-changing behavior based on ESIPT between
hydroxyl and aldehyde groups. It was not only a new breakthrough in anti-counterfeiting and
−
optical imaging, but an efficient fluorescent probe for ClO . Moreover, this result will facilitate the
development of smarter systems and functional compounds that integrate multiple switchable
functions into a single molecule.
2
2
Experimental
.1. Materials and Instrument
Reagents and solvents involved in the experiments were purchased online (AR grade) and there was
no further purification unless mentioned. The use of deionized water was default in all aqueous
1
13
operations and sample preparation. H NMR and C NMR spectra were collected on Bruker-600
MHz and Bruker-400 MHz spectrometer in Deuterated reagents with TMS as an internal standard.
Mass spectra were obtained on a Bruker Ultraflextreme MALDI TOF/TOF mass spectrometer.
UV-vis spectra were recorded on Shimadzu UV-3600 with a UV-VIS-NIR spectrophotometer.
Emission spectra were collected by a HITACHI fluorescence spectrometer (F-4600). The ground
state geometries of all molecules were fully optimized using density functional theory (DFT) at the
B3LYP/6-31G (d, p) level, as implemented in Gaussian 09W software package. The cell imaging
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experiments were carried out by using Confocal Scanning Microscopy (Leica, TCS sp5 II).
2
.2. Synthesis
2
.2.1 Synthesis of 6-bromo-2-butyl-1H-benzo[de]isoquinoline-1,3(2H)-dione (a)
4
-Bromo-1,8-naphthalic anhydride (1.385 g, 5 mmol) and n-butylamine(0.438 g, 6 mmol) were
dissolved in ethanol(30 ml) and refluxed for about 5 hours. The reaction was monitored by TCL
until the end of the reaction. After cooling to room temperature, white product was obtained by
1
recrystallization, 1.494 g. Yield: 90%. H NMR (600 MHz, CDCl ) δ 8.68 (dd, J = 7.3, 0.8 Hz, 1H),
3
8
.58 (dd, J = 8.5, 0.8 Hz, 1H), 8.43 (d, J = 7.8 Hz, 1H), 8.06 (d, J = 7.8 Hz, 1H), 7.87 (dd, J = 8.3, 7.4
Hz, 1H), 4.30 – 4.06 (m, 2H), 1.74 (tt, J = 7.7, 6.7 Hz, 2H), 1.52 – 1.41 (m, 2H), 1.00 (t, J = 7.4 Hz,
1
3
3
H). C NMR (151 MHz, CDCl ) δ 163.65 (d, J = 3.5 Hz), 133.23 (s), 132.03 (s), 131.22 (s), 131.10
3
(s), 130.64 (s), 130.20 (s), 129.02 (s), 128.09 (s), 123.18 (s), 122.31 (s), 40.40 (s), 30.18 (s), 20.39 (s),
+
1
3.86 (s). TOF-MS (ESI) m/z calcd. For C H BrNO [M+H] : 332.0208, found: 332.0200.
1
6
14
2
2
.2.2 Synthesis of 2-butyl-6-methoxy-1H-benzo[de]isoquinoline-1,3(2H)-dione (b)
Compound a (0.996 g, 3 mmol), sodium methoxide (0.648 g, 12 mmol), and CuSO ∙5H O (0.14 g,
4
2
0
.6 mmol) were dissolved in 50ml methanol. The reaction was protected under a nitrogen
atmosphere and was refluxed for about 8 hours to the end. When cooling to room temperature, the
solvent was removed by rotary evaporation and the concentrated product was purified by silica gel
flash column chromatography (dichloromethane (DCM)/hexane (Hex) (1/3, v/v) as the eluent) to get
1
a yellow-green product, 0.64 g. Yield: 75%. H NMR (600 MHz, CDCl ) δ 8.60 (d, J = 7.2 Hz, 1H),
3
8
4
.55 (dd, J = 8.3, 2.0 Hz, 2H), 7.74 – 7.65 (m, 1H), 7.04 (d, J = 8.3 Hz, 1H), 4.20 – 4.16 (m, 2H),
1
3
.14 (s, 3H), 1.77 – 1.65 (m, 3H), 1.51 – 1.42 (m, 2H), 0.99 (t, J = 7.4 Hz, 3H). C NMR (151 MHz,
CDCl ) δ 164.57 (s), 164.02 (s), 160.78 (s), 133.42 (s), 131.53 (s), 129.39 (s), 128.59 (s), 125.95 (s),
3
1
23.51 (s), 122.50 (s), 115.20 (s), 105.18 (s), 56.20 (s), 40.12 (s), 30.29 (s), 20.43 (s), 13.89 (s).
+
TOF-MS (ESI) m/z calcd. For C H NO [M+H] : 284.1208, found: 284.1233.
1
7
17
3
2
.2.3 Synthesis of 2-butyl-6-hydroxy-1H-benzo[de]isoquinoline-1,3(2H)-dione (c)
Compound b (0.566 g, 2 mmol) was added into 20 ml hydro-bromic acid, and an appropriate
amount of glacial acetic acid was put into the flask to increase the solubility. The reaction was
refluxed for 12 hours under a nitrogen atmosphere and was tracked to the end by TCL. After
cooling, the solution was poured into ice water and filtered. The crude product was purified with
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silica gel column chromatography (dichloromethane (DCM)/hexane (Hex) (2/1, v/v) as the eluent) to
1
obtain a pale green product, 0.269 g. Yield: 47.5%. H NMR (600 MHz, DMSO) δ 11.87 (s, 1H),
8
2
1
.65 – 8.05 (m, 3H), 7.46 (ddd, J = 12.7, 11.4, 6.0 Hz, 2H), 3.35 (s, 2H), 1.60 (dd, J = 14.2, 6.7 Hz,
1
3
H), 1.41 – 1.16 (m, 2H), 1.11 – 0.64 (m, 3H). C NMR (151 MHz, DMSO) δ 164.17 (s), 163.50 (s),
60.72 (s), 134.06 (s), 131.64 (s), 129.67 (s), 129.38 (s), 126.13 (s), 122.87 (s), 122.31 (s), 113.11 (s),
1
10.45 (s), 55.39 (s), 30.23 (s), 20.29 (s), 14.21 (s). TOF-MS (ESI) m/z calcd. For C H NO
16 15 3
+
[
M+H] : 270.1052, found:270.1010.
.2.4 Synthesis of 2-butyl-6-hydroxy-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline
5-carbaldehyde (BHC)
2
-
Compound c (269 mg, 1 mmol) and magnesium chloride (140.85 mg, 1.5 mmol) were dissolved in
acetonitrile (15 ml), trimethylamine (404 mg, 4 mmol) was dropped during stirring, and
paraformaldehyde (6.8mmol) was added in the heating process. The reaction was refluxed for 4 h
and was monitored by TCL until finished completely. The mixture solution was adjusted to weak
acidity with 5% dilute hydrochloric acid and the solution was extracted with DCM several times.
The combined organic extracts were dried with MgSO , then filtered, concentrated under vacuum.
4
The residue was purified with silica gel column chromatography (dichloromethane (DCM)/hexane
1
(
Hex) (1:1, v/v) as the eluent), and 195 mg product was collected. Yield: 65%. H NMR (600 MHz,
CDCl ) δ 13.21 (s, 1H), 10.46 – 9.73 (m, 1H), 8.76 (dd, J = 8.8, 7.2 Hz, 3H), 7.84 (t, J = 7.8 Hz, 1H),
3
1
3
4
1
1
.44 – 3.77 (m, 2H), 1.91 – 1.24 (m, 5H), 1.00 (t, J = 7.4 Hz, 3H). C NMR (151 MHz, CDCl ) δ
3
96.47 (s), 165.77 (s), 163.76 (s), 163.10 (s), 134.94 (s), 134.17 (s), 131.86 (s), 130.40 (s), 127.08 (s),
22.92 (d, J = 8.3 Hz), 115.07 (d, J = 36.8 Hz), 53.45 (s), 40.33 (s), 30.21 (s), 20.38 (s), 13.86
+
(
s).TOF-MS (ESI) m/z calcd. For C H NO [M+H] : 298.1001, found: 298.1013.
1
7
15
4
2
.3 The preparation of various analytes
A stock solution of BHC (1.0 mM) was prepared in tetrahydrofuran (THF). The working standard
solutions were prepared by diluting the stock solution to 10 μM by PBS buffer (pH = 7.4). The
sodium hypochlorite (NaClO) solution was prepared by diluting the 14% commercially purchased
NaClO, hydrogen peroxide (H O ) was prepared by diluting the 35% (w/w) purchased one, and all
2
2
ions are obtained by dissolving the corresponding salt in distilled water.
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2
.4 Cell cultures
HeLa cells were cultured on glass-bottom Dulbecco's modified eagle media (DMEM) culture dishes
-1
which were supplemented with 10% (v/v) fetal bovine serum (FBS). 100μg mL
penicillin–streptomycin and were set in a humidified 37 °C incubator containing 5% CO . For the
2
−
detection of exogenous ClO , the HeLa cells were incubated with BHC (10 μM) for 30 mins at
−
3
7 °C and washed with PBS buffer three times, and then added fresh prepared ClO (5 μM) solution
−
in culture media and stay for 30 mins. For the detection of endogenously produced ClO , the HeLa
cells were incubated with BHC (10 μM) for 30 mins at 37 °C incubator, and then washed with PBS
buffer three times before being stimulated by 2 ug/mL lipopolysaccharides (LPS) and phorbol
1
2-myristate 13acetate (PMA) for 2 hours. As a control experiment group, the HeLa cells were
incubated with BHC (10 μM) for 30 mins at 37 °C incubator, and then washed with PBS buffer
three times, then stimulated with 200 μM 4-aminobenzoic-acid-hydrazide(ABH) and 2 ug/mL
lipopolysaccharides (LPS) and phorbol 12-myristate 13 acetate (PMA) for 2 hours.
3
3
. Results and discussions
.1 Synthesis and characterization
The synthetic procedure for compound BHC was illustrated in Scheme 1 using
-Bromo-1,8-naphthalic anhydride as the starting material. Firstly, compound a was conveniently
4
prepared in high yield in ethanol. Then, intermediate a was effectively transformed into b in the
presence of CH ONa, CuSO ∙5H O in methanol under N protection. Third, compound c was
3
4
2
2
directly synthesized in a high yield by a simple reduction reaction. At last, compound c reacted with
the paraformaldehyde in acetonitrile to give the BHC, which was a new method used in this article.
The raw BHC was purified through column chromatography (Yield : 65%) and was structurally
1
13
characterized by H-NMR, C-NMR, and MALDI TOF/TOF mass spectrometer techniques. In a
word, compound BHC had been efficiently synthesized in relatively high yield and a new and
simple synthetic method
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Scheme 1 Synthetic routes of compound BHC
3
3
.2 Photochromic properties
.2.1 The reversible photochromic properties of BHC
The absorption and photoluminescence (PL) spectra of the solid BHC before and after UV
irradiated were recorded in Fig.1. It could be seen that the absorption of BHC after UV irradiation
was remarkably enhanced between 425 nm and 475 nm, which means an extension of the
conjugated structure caused by the change of the enol to the trans-keto structure. There were two
emissions in the PL spectrum, one at 462 nm and the other at 546 nm. After intense ultraviolet
excitation at 390 nm, a sharp decrease in the fluorescence intensity at 464 nm were observed in
Fig.1b. The former was the fluorescence of BHC itself and the latter might caused by the excited
state intramolecular proton transfer of some compounds (ESIPT). Under excitation at 390 nm, most
of the compounds converted to the trans-keto structure, and intramolecular push-pull electron effect
was greatly attenuated while electron-withdrawing ability of the ketone structure was prominent.
The initial state of BHC exists in the structure of enol and after being excited with higher energy
ultraviolet light, proton transferred from hydroxyl to aldehyde (Scheme 2). Such movement is
reversible, and the initial enol can be achieved again under long wavelengths of light and heating
condition. The UV absorption and fluorescence spectra were mutually confirmed, indicating the fact
that BHC has experienced the process from an enol form to a trans-keto one.
(a)
(b)
Fig. 1. (a) The UV–vis absorption spectra of BHC before and after UV irradiation in solid state (b) The PL spectra
of of BHC before and after UV irradiation in solid state (λ = 390 nm, λem = 462 nm, 546 nm, slit: 5 nm/5 nm).
ex
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Scheme 2 Photochromic mechanism of BHC upon UV irradiation
In addition to the above photochromic properties, the solid trans-keto compounds also exhibited
thermal fading and photo bleaching properties, and the results are shown in the Fig.2. In daylight,
the white compound turns yellow after exposure to ultraviolet light for 20 seconds and then returns
to milky white when heating or irradiate with intense white light. At the same time, the fluorescence
changes from light blue to deep yellow, again returning to the initial state under the 'washing' of
heat and light. The compound not only shows a time-dependent fading rate, but also exhibits
thermal fading ability. Specifically, the rate of fading is positively correlated with temperature. As
shown in the Fig.2b, the slope of the fitted line, absorption intensity at 409 nm as the ordinate and
time as the abscissa, is -0.00607 for 65 ℃, which is twice of that of 25 ℃ (0.00392). The data under
2
5 ℃, 45 ℃, 65 ℃ fully demonstrate the relationship between fading rate and temperatures, which
has a great guiding effect on the application range of compound (Fig.S2).
(a)
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1
1
0
0
0
0
0
.2
.0
.8
.6
.4
.2
.0
0
0
0
.30
.29
.28
F= -0.00607 [t] + 0.294
2
R = 0.992 [t] = 0-6 mins
0.27
.26
0.25
0
0
2
4
6
8
10
Time(mins)
3
60
390
420
450
480
510
540
Wavelength (nm)
(b)
Fig. 2. (a) Photographs of BHC before and after photochromism, and photos after long-wavelength illumination
or heating. (b) The thermal fading kinetics of BHC at 65 ℃.
3
.2.2 Theoretical calculation
DFT calculations of BHC Enol and trans-Keto were performed on the B3LYP/6-31G* level to gain
a deeper understanding of the photochromic properties (Fig. 3a). Theoretically, the energy gap (EG)
between HOMO and LUMO was related to the absorption wavelength: a smaller EG was conducive
to electron transport, causing a red-shift in absorption wavelength. As expected, Enol's EG was
3
.778 ev and trans- Keto's EG was 3.416 ev (Fig. 3a), which was very consistent with the
red-shifted absorption wavelength in Fig. 1a. The trend that HOMO and LUMO of trans-keto
shifting to the carbonyl moiety confirmed the electron-withdrawing property of the carbonyl moiety.
It also suggested that BHC photochromism was caused by the absorption of energy from UV
irradiation. As a photochromic material, its fatigue resistance and erasability were properties that
need to be studied. The fatigue resistance of BHC was investigated by repeatedly switching light
conditions for five times. As shown in the Fig.3b, the fluorescence remained almost constant
without degradation, and the pattern written on the tiled compound by a laser pointer intuitively
reflected discoloration process, indicating good fatigue resistance of BHC. Reversible
photochromic properties and good fatigue resistance, small degree of color change indicated its
potential to become an novel anti-counterfeiting material with higher secrecy coefficient or
decorative material, and its commercial production application is promising.
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LUMO
HOMO

E=3.778 ev
Enol
-2.865 ev
-6.643 ev

E=3.416 ev
trans-Keto
-2.891 ev
-6.307 ev
(a)
(b)
Fig.3. (a) Frontier molecular orbital amplitude plots and energy levels of the HOMOs and the LUMOs of BHC
Enol and trans-Keto. (b)The modulation of the FL intensity of BHC at 546 nm as a result of alternating irradiation
and heating treatment.
−
3
3
.3 Hypochlorite (ClO ) Detection properties
.3.1 Photoluminescence and UV–vis spectra in water solution
The UV absorption and photoluminescence (PL) spectra of the compound in a complete PBS
solution were shown in Fig.4a. The maximum absorption is around 409 nm and the maximum
emission is around 510 nm, which results in strong yellow-green fluorescence (365nm handheld UV
light) and a large Stokes shift. The PL quantum yield of the compound BHC is as high as 32.06%
which is a relatively higher PL quantum yield compared to other water-soluble dyes. As shown in
Fig. 4b, with sodium hypochlorite solution dropped, the PL of solution quenched from bright green
to colorless, which facilitates visual inspection of the test results.
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(a)
(b)
Fig.4. (a) The UV–vis absorption spectra and photoluminescence (PL) spectra of BHC (10 μM) in PBS buffer
−
solution (pH 7.4). (b) The PL spectra of probe BHC (10 μM) in the presence of ClO (50 μM) in PBS buffer
solution (pH 7.4) (λ = 409 nm, λ = 510 nm, slit: 5 nm/5 nm).
ex
em
3
.3.2 Selectivity
In order to evaluate the hypochlorite selectivity of the probe, the photoluminescence (PL) spectra of
PBS buffered solution containing the probe and the analytes (100 equiv.) were recorded in Fig.5 and
were used to study the possible effects of other analytes posed on the probe. The results showed that
2
+
2+
+
2+
+
2+
−
−
−
−
−
−
−
−
all the analytes such as Ba , Ca , K , Mg , Na , Zn , Br , Cl , ClO , F , I , HS , NO , NO ,
4
2
3
2−
−
6
−
−
−
SO , H O , PF , CN , AcO did not significantly shade the PL of the solution. When ClO was
4
2
2
added, the PL quenched quickly. Moreover, for the purpose of exploring the effect of the probe on
−
−
the competitive recognition of ClO , ClO being added into the mixed solution in the presence of
multiple ions, the results showed that the recognition effect was still quite optimistic (as shown in
−
Fig. 5b). A good selectivity for ClO among various analytes were exhibited through above
experiments.
(
a)
Fig.5. (a), (b) The photoluminescence (PL) spectra of probe (10 μM) with various analytes (50 μM for ClO and
00 μM for other analytes) in PBS buffer solution (λex = 409 nm, λem = 510 nm, slit: 5 nm/5 nm pH 7.4).
(b)
−
1
3
.3.3 pH dependent
−
The effect of different pH on the identification of ClO was analyzed. Photoluminescence (PL)
−
intensity of the probe and probe with ClO at different pH conditions were recorded in Fig.6. In the
−
pH range of 1-4, the PL of probe is very weak. As is known to all, ClO is not stable in strongly
acidic solutions. Therefore, the detection results in the pH range of 1-4 are negligible. When pH
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−
exceeds 10, the OH in the alkaline solution largely hinders direct contact between protons on the
−
hydroxyl group and ClO and the solution only shows the photoluminescence (PL) of the probe.
Therefore, the probe covers the physiological pH range (5-9), which brings great application
prospects.
−
Fig.6. Effect of the pH on the photoluminescence (PL) intensity of probe alone and probe reacted with ClO (10
equiv) at different pH (4.5-9.5) (λex = 409 nm, λem = 510 nm, slit: 5 nm/5 nm).
−
3
.3.4 The detection limit of probe for ClO
-8
The detection limit of the probe is 1.16×10 M, which was calculated by the formula:
Detection limit=3σ/k, where σ is the standard deviation of blank sample, k is the slope between the
−
fluorescence intensity (I510 nm) and the concentration of ClO . The fluorescence emission spectra of
the probe (10 μM) were measured 11 times to obtain the standard deviation of the blank sample.
−
The fluorescence spectra of probe with different equivalent amounts of ClO (0 eq-10 eq) was
recorded 20 seconds after addition (Fig.7a) and the fluorescence intensity at 510 nm was taken as
−
the ordinate, and the concentration of ClO was plotted on the abscissa, fitting a straight line (Fig.
7
b).
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(a)
(b)
−
Fig.7. (a) The photoluminescence (PL) spectra of probe (10 μM) in the presence of various concentrations of ClO
(0-10 eq) in PBS buffer solution (λex = 409 nm, λem = 510 nm, slit: 5 nm/5 nm). (b) Plot of the
−
photoluminescence (PL) intensity of probe at 510 nm as a function of the concentration of ClO (λex = 409 nm).
3
.3.5 Proposed mechanism
1
Based on reasonable and reliable characterization of H NMR, the probe detection mechanism was
as shown in Figure.8. The oxidation of hypochlorous acid prompts us to conjecture that the phenolic
hydroxyl group was oxidized to aryl ketone, therefore, a crude color reaction were performed. An
aqueous solution of ferric chloride was added to the probe solution with no hypochlorous acid and
the solution was dark purple-red. However, when ferric chloride solution was added to the solution
to which sodium hypochlorite had been added before, there was no noticeable discoloration, which
initially demonstrated conversion of the phenolic hydroxyl group. In order to figure out exact
recognition mechanism of the probe, hypochlorous acid was titrated to deuterated reagents with
1
dissolved probes and H NMR shows an obvious disappearance of phenolic hydroxyls ( Fig.S3†.).
This indicates that hypochlorite oxidation probes only oxidize hydroxyl groups to carbonyl groups.
−
Fig.8. The proposed mechanism of the probe BHC sensing ClO .
3
.3.6 Cellular imaging
Considering the excellent water solubility and bright fluorescence as well as the recognition for
−
−
ClO , the potential applications in the field of cell imaging and exogenous ClO detection were
further explored. Fluorescence imaging experiments were performed in living HeLa cells who were
set on 6-well plates with BHC and adhere for 24 hours. The cells were washed three times with
PBS buffer solution before the experiment. Firstly, four groups of cells were incubated with 10 μM
BHC aqueous solution in a 37 °C incubator for 30 minutes and then washed three times with PBS
buffer solution. When the probe alone incubated the cells, they presented bright fluorescence under
the green channel (as shown in Fig.9.A). However, when exogenous hypochlorous salt was added to
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the medium for 30 minutes, the fluorescence disappeared because the probe reacted with
hypochlorous acid and Intramolecular charge transfer mechanism in molecule was broken (as
shown in Fig.9.B). In the process of tracking cell endogenous hypochlorous acid, pretreated cells
were stimulated with lipopolysaccharides (LPS) and phorbol 12-myristate 13 acetate (PMA) to give
some hypochlorous acid. Similarly, it was observed that the fluorescence of the cells was shut down,
−
indicating that the probe is able to monitor ClO successfully (as shown in Fig.9.C). As a
comparative experiment, aside from LPS and PMA, ABH was added to inhibit the production of
hypochlorous acid in cells and it was found that the fluorescence of the cells did not disappear (as
shown in Fig.9.D).
Fig.9. Confocal fluorescent image of HeLa cells. (A1-3) Fluorescent image of cells incubated with probe (10 μM)
for 30 mins under bright field, green channel and the merge respectively. (B1-3) Fluorescent image of cells treated
−
with probe (10 μM) and then treated with ClO (5 μM) for 30 mins. (C1-3) Fluorescent image of cells treated with
probe (10 μM) and then treated with LPS and PMA(2 ug/mL) for 30 mins under bright field, green channel and
the merge respectively. (D-3) Fluorescent image of cells treated with probe (10 μM) and then treated with ABH,
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LPS and PMA(2 ug/mL) for 30 mins under bright field, green channel and the merge respectively.
At the same time, the toxicity and photoluminescence (PL) stability of the probe in cells were
discussed. After 8 hours of incubation with different concentrations of probe, cell survival rate
remained high and the photoluminescence intensity within 8 minutes was also relatively stable
(Fig.10). Therefore, from the above analysis and experiments, it could be seen that the probe can
not only stain cells, but also accurately track the generation of hypochlorous acid in vivo and in
vitro, thus broadening the path for cell imaging and biological applications.
(a)
(b)
Fig.10. (a) The survival rate of cells cultured with different probe concentrations (0-30 μm) for 8 hours. (b)
Stained cell photoluminescence (PL) intensity versus time in 8 mins.
4
. Conclusion
In summary, a bifunctional, water-soluble BHC has been synthesized, presenting a photoswitch
−
ability that responding to ClO and alternating UV/vis light irradiation. BHC shows a reversible
photochromic behavior, good fatigue resistance and adjustable bleaching rate, which broadens
choice of smart switching and anti-counterfeiting marking materials. The selectivity to hypochlorite
−
at physiological pH makes it suitable for cell imaging and ClO detection in vitro and vivo. It has
been proved non-toxic by cell cultivation test and bright green to no fluorescence detection result is
easy to identify. The practical applications of probe such as imaging living cells and the detecting of
hypochlorite in vivo were confirmed by cell culture experiments. The detection limit was as low as
-8
1
.16×10 M. This dual functional compound is not only a new breakthrough in anti-counterfeiting
−
and optical imaging, but also a practical fluorescent probe for ClO , which provides evidence for
the idea of integrating multiple functions into one molecule.
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Acknowledgements
This work was supported by the Fundamental Research Funds for the Central Universities
2019XKQYMS72) and the Priority Academic Program Development of Jiangsu Higher Education
(
Institutions.
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Graphical abstract
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Highlights


This is an interesting naphthalimide-based bifunctional material with reversible
photochromic behavior and ClO recognition effect.
The compound BHC exhibits a rare naphthalimide-based ESIPT mechanism for
photochromic properties and fatigue resistance.
−

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This is a completely water soluble fluorescent probe
The probe BHC is capable of simultaneously detecting ClO in aqueous solution
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and living cells with high sensitivity and selectivity, and exhibits obvious "on-off"
fluorescence.
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Probe BHC exhibited ideal properties including high sensitive, good selectivity
and short response time.
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