AIE-based fluorescent boronate probe and its application in peroxynitrite imaging

Article abstract of DOI:10.1016/j.saa.2021.120044

Fluorescent probes have contributed greatly to our understanding of the biological role of peroxynitrite (ONOO^–). The ONOO^– fluorescence probe characterized by the arlyboronate received a moderate opening fluorescence response, and t

Full text of DOI:10.1016/j.saa.2021.120044

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 261 (2021) 120044
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Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
AIE-based fluorescent boronate probe and its application in peroxynitrite
imaging
a
b,
Minglu Li a, Hui Han b, Shengmei Song b, , Shaomin Shuang , Chuan Dong
⇑
⇑
a School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, PR China
b Institute of Environmental Science, Shanxi University, Taiyuan 030006, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
ADB with aggregation-induced
emission process exhibited good
sensitivity for ONOO–.
ꢀ
Emission wavelength of probe was
affected by the electron donating (or
withdrawing) of the substituents.
ADB can detect ONOO– rapidly (20 s).
ADB displayed good performances for
exogenous ONOO– in the RAW 264.7
cells.
ꢀ
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
Fluorescent probes have contributed greatly to our understanding of the biological role of peroxynitrite
(ONOO–). The ONOO– fluorescence probe characterized by the arlyboronate received a moderate opening
fluorescence response, and the borate-masked probe significantly increased the sensitivity of ONOO–.
Thus, two simple fluorescent probes (ADB and ANB) with the recognition receptor of phenyl boronate
moiety were constructed for the detection of ONOO–. The change of emission spectrum was affected dif-
ferently by the electron donating (or withdrawing) of the substituents. ANB was shown to have a low
sensitivity and quantum yield towards ONOO– in aqueous solution, whereas ADB with aggregation-
induced emission (AIE) process exhibited not only good sensitivity for ONOO– with a detection limit of
75 nM, but also ADB could be used to quantitative detecting ONOO– in response to concentrations of
ONOO– within 20 s. Importantly, ADB had good performance for the detection of exogenous ONOO– in
the RAW 264.7 cells.
Received 6 March 2021
Received in revised form 12 May 2021
Accepted 31 May 2021
Available online 4 June 2021
Keywords:
Fluorescent probes
Peroxynitrite
Arlyboronate
AIE
Ó 2021 Elsevier B.V. All rights reserved.
1. Introduction
including proteins [2] and DNA [3], which may damage critical cell
components and function, and induced various diseases including
Peroxynitrite (ONOO–), refered to as a highly reactive species,
was formed from the immediate combination of superoxide (O–2ꢁ)
and nitric oxide (NO.) in the biological systems. [1] Excessive
ONOO– may damage multiple molecular components in the cells,
cancer [4], Alzheimer’s disease [5], inflammatory diseases [6] and
so on. In addition, ONOO– was difficult to measure in biological
systems due to it being short half-life (about 1 s) and steady-
state concentration of nanomolar range in the cells [7,8]. Thus,
the importance of ONOO– have captured the attention of many
researchers who seek selective and sensitive approaches for
ONOO–. Currently, the techniques for detecting changes in ONOO–
⇑
Corresponding authors.
E-mail addresses: smsong@sxu.edu.cn (S. Song), dc@sxu.edu.cn (C. Dong).
https://doi.org/10.1016/j.saa.2021.120044
1386-1425/Ó 2021 Elsevier B.V. All rights reserved.

M. Li, H. Han, S. Song et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 261 (2021) 120044
concentrations in biological systems include spectroscopy [9], elec-
trochemistry [10], electron spin resonance etc [11]. Among them,
the fluorescence approach has been developed as a powerful tool
for ONOO– in terms of simplicity, sensitivity, real-time and non-
destructive detection in living systems [12–16].
mass spectra were obtained on a Q Exactive mass spectrometer
(Thermos Fisher Scientific). The pH was adjusted using a Phs-3c
pH meter (Germany Sartorius).
2.2. The preparation of various analytes
Consequently, numbers of small-molecular fluorescent probes
for ONOO– have been developed in the past several years,[17–26]
especially aryboronate-based fluorescent probes [21–22]. For
example, Chang et al. proposed the borate ester fluorescent probes
for hydrogen peroxide (H2O2) [27]. As a result of the progress of
ONOO– research, in 2009, it was reported firstly that ONOO– and
H2O2 had a similar reaction with boric acid ester group to form
phenol, but the reaction rate with ONOO– was faster [28]. Han
et al. used pyrene fluorophores as chromophores to design and
synthesize fluorescent probes [29]. Nevertheless, there was consid-
erable challenges such as its coexistence with other reactive oxy-
gen species, especially H2O2 in biological systems due to
structural similarity. In addition, low water solubility will limit
its application in biological systems. Qian et al. constructed a fluo-
rescence on–off probe, which destroyed the interaction between B
and N atoms in the probe by the reaction of ONOO– with
fluorescence-closed photoinduced electron transfer probe [30].
Moreover, the probe had the advantage of not being interfered
by H2O2 and reacting rapidly (1 min) with ONOO–, the disadvan-
tage was that the probe and HClO can also cause significant fluo-
rescence attenuation. Therefore, certain aspects still need to be
improved in sensitivity, selectivity and response speed.
It was known that benzothiazole derivatives have inherent
advantages of simple synthesis, easily available raw materials,
good photostability and ratiometric sensing, providing a fluo-
rophore with excellent fluorescence performance [31–32]. View
of this, 2- (20-hydroxyphenyl) benzothiazole was selected as the
framework to design and synthesize aryboronate fluorescent
probes ADB and ANB for ONOO–. Compared to ANB, ADB exhibited
displayed a good linear range of 0.15–3.9 µM and low detection
limit of 75 nM, fast response for ONOO– would help track the
highly reactive and short-lived ONOO– in living cells. In addition,
the probe displayed good performance in detecting exogenous
ONOO– and had great potential in practical applications of biolog-
ical systems.
We prepared reactive oxygen species as follows:
H2O2: Dilution of a 30% solution in deionized water to obtain
H2O2, whose concentration was determined using extinction coef-
ficient of 43.6 Mꢂ1cmꢂ1 at 204 nm. NO: Sodium (III) nitroferri-
cyanide dihydrate was dissolved in deoxygenated ultrapure
water and stored at 4 °C to prepare NO. ClO-: Dilution of 5% NaClO
solution in deionized water to generate ClO-, whose concentration
was obtained using extinction coefficient of 350 Mꢂ1cmꢂ1 at
292 nm. 1O2: The reaction of ClO- and H2O2 to obtain 1O2. Oꢁ2-:
KO2 was dissolved in dry DMSO to prepare superoxide. ꢁOH: The
Fenton reaction of Fe2+ and H2O2 to generate ꢁOH. NO–2: Dissolving
NaNO2 in ultrapure water to prepare NO–2. ONOO–: ONOO– was
synthesized by the reaction of NaNO2 and H2O2 under concen-
trated hydrochloric acid condition and stored in sodium hydroxide
solution, and concentration of ONOO– was determined by UV–Vis
spectrophotometer (e
was 1670 Mꢂ1 cmꢂ1 at 302 nm).
2.3. Synthesis of AD
A mixture of 2-aminothiophenol (0.52 g, 4.2 mmol) and 4-
(diehylamino) salicylaldehyde (0.6 g, 3.2 mmol) was dissolved in
ethanol (10 mL), then hydrochloric acid (0.79 mL, 37%) and H2O2
(1.93 mL, 30%) were added to the mixture solution and stirred
for 90 min at room temperature, the reaction was quenched with
10 mL water, filtered and recrystallized using ethanol to afford yel-
low solid (0.7 g, 74%). FT-IR (
m m (O-H): 3438; m (C-H):
/cmꢂ1):
2973; (C = N): 1632;
m
m
(C = C): 1560, 1469, 1429. 1H NMR
(600 MHz, DMSO d6, ppm) d 11.73 (s, 1H), 8.03 (s, J = 7.5 Hz, 1H),
7.90 (d, J = 7.9 Hz, 1H), 7.71 (t, J = 8.1 Hz, 1H), 7.47 (t, 1H), 7.34
(d, 1H), 6.38 (d, J = 8.2 Hz, 1H), 6.21 (d, 1H), 3.38 (q, 4H), 1.13 (t,
6H). 13C NMR (151 MHz, DMSO d6, ppm)
d
167.89, 159.04,
152.16, 151.45, 132.82, 130.34, 126.83, 124.61, 122.23, 121.24,
106.09, 104.98, 97.59, 44.38, 13.02. HRMS (ESI): Calcd for C17H18
-
+
N2OS [AD + H] 299.1218, found 299.12155.
2.4. Synthesis of ADB
2. Experimental
Intermediates AD (0.11 g, 0.36 mmol) and K2CO3 (0.12 g,
0.86 mmol) were dissolved in 5 mL DMF at room temperature,
then 4-bromomethylphenylboronic acid pinacol ester (0.127 g,
0.43 mmol) was added to above mixture solution and stirred 5 h
at 60 °C, the residue was concentrated in vacuo. The obtained solid
was purified using silica gel chromatography with dichloro-
methane and methanol (10:1, v/v) as eluent to afford ADB as a
solid (0.098 g, 53%). The melting points of ADB: 138.9 °C. FT-IR
2.1. Materials and apparatus
4-bromomethylphenylboronic acid pinacol ester, ebselen, 2-
aminothiophenol, 4-(diehylamino) salicylaldehyde and 5-
Nitrosalicylaldehyde were purchased from Aladdin (Shanghai,
china). 3-morpholino sydnonimine hydrochloride (SIN-1) was pur-
chased from Toronto Research Chemicals INC (Canada). K2CO3 was
brought from Macklin Biochemical Company (Shanghai, china). All
chemistry reagents are analytical grade without further purifica-
tion. All aqueous solutions were prepared using deionized water
from a Milli-Q water purification system (Millipore, Bedford, MA,
USA).
(m m (C-H): 2974; m (C = N): 1608; m (C = C): 1554, 1521,
/cmꢂ1):
1461. 1H NMR (600 MHz, DMSO d6, ppm) d 8.66 (s, 1H), 8.22 (d,
J = 8.7 Hz, 1H), 7.93 (t, J = 9.4 Hz, 1H), 7.72 (t, J = 9.0 Hz, 1H), 7.49
(d, J = 7.8 Hz, 2H), 7.31 (d, 1H), 7.22 (d, 1H), 5.75 (d, 2H), 5.29 (d,
1H), 3.69 (s, 2H), 3.32 (t, 2H), 3.18 (t, J = 28.5 Hz, 1H), 3.04 (s,
1H), 1.27 (s, J = 22.6 Hz, 13H), 1.06 (t, 5H). 13C NMR (151 MHz,
DMSO d6, ppm) d 168.82, 168.26, 163.26, 160.33, 150.36, 144.73,
139.96, 139.82, 132.31, 132.19, 125.66, 124.49, 123.09, 88.96,
Absorption spectra were measured on a Lambda 365 spec-
trophotometer (PerkinElmer, U.K.). Fluorescence spectra were per-
formed on
a spectrophotometer (Varian Cary Eclipse). The
fluorescence emissions of AD in CH3CN/water mixed solutions
were carried out on a FLS-920 Edinburgh Fluorescence Spectropho-
tometer (Edinburgh Co., Ltd., England). Fourier transform infrared
(FTIR) spectrometer (Bruker Tensor Ⅱ) was recorded with KBr pel-
let in order to analyze the information of chemical bonding. 1H
NMR and 1C NMR spectra were carried out on an AVANCE-
600 MHz NMR Spectrometer (Germany Bruker). High-resolution
78.73, 74.90, 60.15, 50.57, 29.90, 17.66. HRMS (ESI): Calcd for C30-
+
H35BN2O3S [ADB + H] 515.2540, found 515.25382.
2.5. Synthesis of AN
A mixture of 2-aminothiophenol (0.52 g, 4.2 mmol) and 5-
Nitrosalicylaldehyde (0.535 g, 3.2 mmol) was dissolved in ethanol
2

M. Li, H. Han, S. Song et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 261 (2021) 120044
(10 mL), then hydrochloric acid (0.79 mL, 37%) and H2O2 (1.93 mL,
30%) were added to the mixture solution and stirred for 90 min at
room temperature, the reaction was quenched with 10 mL water,
filtered and recrystallized using ethanol to afford yellow solid
(0.67 g, 78%). 1H NMR (600 MHz, DMSO d6, ppm) d 12.97 (s, 1H),
9.16 (d, 1H), 8.27 (d, J = 21.1, 7.1 Hz, 1H), 8.14 (d, J = 26.3, 18.3,
8.0 Hz, 2H), 7.57 (t, J = 12.3, 7.4 Hz, 1H), 7.48 (t, 1H), 7.24 (d, 1H).
13C NMR (151 MHz, DMSO d6, ppm) d 161.77, 161.65, 151.73,
140.47, 135.83, 127.68, 127.10, 125.87, 124.80, 123.14, 122.60,
terization of ADB and ANB were presented in the supporting infor-
mation (Fig. S9-S22). To construct the specific and selective
recognition receptors of ONOO–, we employed benzothiazole as
fluorophores, the chemical transformation of aryboronate to phe-
nol was selected as the reaction site. Arylboronate-based fluores-
cent probes had been used to detect H2O2 in living cells.
Arylboronate as an excellent reaction unit could rapidly react with
ONOO– to obtain hydroxyl derivatives. In addition, ONOO– should
be a nucleophile that the reaction of ONOO– with arylboronate
was easier than that of H2O2 to undergo subsequent aryl migration
reaction, because NO–2 was a better leaving group than OH–.[33]
Therefore, arylboronate-based fluorescent probes were very
promising to realize the rapid and specific response toward
ONOO–. 2- (20-hydroxyphenyl) benzothiazole derivatives were
regarded as ideal ESIPT fluorophore due to having a free phenol
group (AD and AN), and functionalization of free phenolic unit with
a benzyl boronic ester would block the ESIPT process. As shown in
Fig. 1a, AN exhibited a good solvation effect, and the peaks of emis-
sion spectra were significantly different in solvents of various
polarities. After functionalization of free phenolic unit with a ben-
zyl boronic ester (ANB and ADB), the peaks of emission spectra did
not obvious change in solvents of different polarities and could
block the ESIPT process (Fig. 1b, c).
+
120.13, 118.05. HRMS (ESI): Calcd for C13H8N2O3S [AN + H]
273.0344, found 273.03265.
2.6. Synthesis of ANB
Intermediates AN (0.107 g, 0.36 mmol) and K2CO3 (0.12 g,
0.86 mmol) were dissolved in 5 mL DMF at room temperature,
then 4-bromomethylphenylboronic acid pinacol ester (0.127 g,
0.43 mmol) was added to above mixture solution and stirred 5 h
at 60 °C, the residue was concentrated in vacuo. The obtained solid
was purified using silica gel chromatography with dichloro-
methane and methanol (10:1, v/v) as eluent to afford ANB as a
solid (0.105 g, 60%). The melting points of ANB: 172.7 °C. 1H
NMR (600 MHz, DMSO d6, ppm) d 9.26 (d, 1H), 8.40 (d, J = 9.3 Hz,
1H), 8.17 (d, J = 8.3 Hz, 2H), 7.84 (t, J = 7.5 Hz, 1H), 7.75 (d,
J = 7.4 Hz, 2H), 7.60 (d, 2H), 7.48 (t, J = 7.5 Hz, 1H), 5.66 (d,
J = 15.5 Hz, 1H), 3.95 (s, 2H), 1.28 (s, J = 14.5 Hz, 12H). 13C NMR
3.2. Spectroscopic properties of ADB towards ONOO–
We initially performed the UV–Vis behavior in PBS buffer solu-
tion (20 mM, pH 7.4, 1% DMSO). As shown in Fig. S1a, the main
absorption band of ADB at 362 nm disappeared with a new band
at 378 nm when the successive addition of ONOO–. And as shown
in Fig. S1b, the absorption spectra of ANB at 363 nm appeared
gradually when the successive addition of ONOO–. Next, the fluo-
rescence spectra of ADB and ANB with increasing amount of
ONOO– in PBS buffer solution (20 mM, pH 7.4, 1% DMSO). As shown
in Fig. 2a, ADB itself showed a weak fluorescence emission peak at
440 nm with the quantum yield of 0.02, a dose-dependent fluores-
cence increment was observed after incubation with different con-
centration of ONOO– (0–5.7 µM) with the quantum yield of 0.10,
ADB produced an up to 7.6-fold fluorescence ‘‘turn on” in the pres-
ence of ONOO–, and the intensity at 440 nm exhibited an excellent
linear correlation (R2 = 0.9921) with the added ONOO– concentra-
tion (0.15–3.9 µM). The detection limit was determined to be
(151 MHz, DMSO d6, ppm)
135.24, 134.87, 128.07, 127.71, 127.67, 127.23, 126.12, 124.70,
123.38, 122.62, 122.38, 116.09, 115.22, 84.25, 73.97, 71.86, 25.42,
25.14. HRMS (ESI): Calcd for C26H25BN2O5S [ANB + H] 489.1655,
found 489.16484.
d 151.70, 151.68, 141.72, 135.98,
+
2.7. UV–Vis and fluorescence spectral measurements
For UV–vis titrations, a stock solution of the ADB and ANB was
prepared (1 ꢃ 10ꢂ3 M) in dimethyl sulfoxide and were diluted to
10 lM in PBS buffer solution (2 mL, pH = 7.4, 20 mM) for the spec-
troscopic experiments. The stoke solutions (0.1 M) of the guest
cations like Ca2+, Cd2+, Mn2+, Cu2+, Ni2+, Ba2+, Al3+, Mg2+, Hg2+
,
Cr3+, Ag+, Zn2+ and anions like ClO-4, F-, Cr2O72-, S2O23-, I-, CO32–, NO–3,
S2- were prepared in deionized water. In the all spectroscopic
experiments, the diluted solutions were added into a quartz optical
cell and each optical path length of cell was 1 cm.
75 nM on the basis of the formula of 3r/k (Fig. 2b), which can
achieve a highly sensitive detection of ONOO–. The fluorescence
variation of ADB was attributed to ONOO–-triggered oxidative
reaction. As shown in Fig. 2c, ANB itself showed a weak fluorescent
emission peak at 520 nm with the quantum yield of 0.001, and the
emission peak of ANB at 520 nm decreased gradually with the
addition of increased amount of ONOO– due to the donor-excited
photoinduced-electron-transfer process from the excited fluo-
rophore to the electron-deficient nitro-substituted benzene moi-
ety. As depicted in Fig. 2d, the intensity of ANB at 520 nm
exhibited a poor linear correlation (R2 = 0.9631) over the added
ONOO– (0.15–0.75 µM). Because of ANB had a low sensitivity and
quantum yield towards ONOO– in aqueous solution, we turned
our attention towards the evaluation of other properties of ADB.
2.8. Cell culture and confocal microscopy
Raw 264.7 cells were cultured in Dulbecco’s Modified Eagle
Medium (DMEM) with 10% FBS, 100 µg/ml streptomycin, and 100
units/ml penicillin G. The cells were cultured in a humidified atmo-
sphere containing 5% CO2 at 37 °C. For monitoring of ONOO– in the
RAW 264.7 macrophage cells, the cells were cultured with 50 µM,
200 µM and 500 µM SIN-1 for 1 h and washed three times with
PBS. Then the RAW 264.7 cells were cultured with 10 µM ADB
for 10 min at 37 °C and washed with PBS for 3 times. The fluores-
cence images were recorded with Olympus FV1000 confocal
microscope (Tokyo, Japan).
3.3. Selectivity studies, pH effect and the response of time-dependence
3. Result and discussion
Next, the specificity of ADB was examined by evaluating fluo-
rescence response (Fig. 3a, b). As expected, ADB achieved signifi-
cant fluorescence enhancement only in the presence of ONOO–,
and ADB exhibited negligible fluorescence with 100 µM of interfer-
3.1. Rational design of probe
At present, fluorescent probes with various reaction mecha-
nisms are emerging (Table S1), we designed and synthesized
arylboronate-based fluorescent probes ADB and ANB, the synthetic
routes were shown in Scheme 1, the detailed synthesis and charac-
ing species (Na+, Ca2+, Cd2+, Mn2+, Cu2+, Ni2+, Ba2+, Al3+, Mg2+, Hg2+
,
Cr3+, Ag+, Zn2+, ClO4- , F-, Cr2O72-, S2O23-, I-, CO23–, NO–3, S2-, ClO-, H2O2,
1O2, NO, Oꢁ2-, ONOO–), which suggested ADB had excellent selectiv-
3

M. Li, H. Han, S. Song et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 261 (2021) 120044
Scheme 1. Preparation of ADB and ANB.
Fig. 1. Fluorescence spectra of AN (a), ANB (b) and ADB (c) in different solvents.
Fig. 2. (a) Fluorescence response of ADB (10 µM) with different concentrations of ONOO– (0–5.7 µM). (b) Linear correlation between the fluorescence intensity of ADB at
440 nm and ONOO– concentration (0.15–3.9 µM), Slit: 5/10. (c) Fluorescence response of ANB (10 µM) with different concentrations of ONOO– (0–0.75 µM). (d) Linear
correlation between the fluorescence intensity of ANB at 520 nm and ONOO– concentration (0.15–0.75 µM), Slit: 10/10.
4

M. Li, H. Han, S. Song et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 261 (2021) 120044
Fig. 3. Fluorescence response (a) and the columns plot (b) of ADB (10 µM, 1% DMSO, pH = 7.4) toward 100 µM analytes (unless otherwise noted). (c) Fluorescence responses
of ADB in the absence and presence of 10 µM ONOO– in the pH range 2.0–12.0. (d) Real-time fluorescence changes of ADB to ONOO– (10 µM).
ity for ONOO– under simulated physiological condition. In addition,
the pH effect on the fluorescence response of ADB to ONOO– was
tested (Fig. 3c). The fluorescence intensity of ADB kept extremely
low in the pH range of 2.0–12.0. After adding 10 µM ONOO–, in
the pH range of 7.0–12.0, the fluorescence intensity increases in
multiples higher than pH 2.0–6.0, due to the fact that ONOO–
was more easily protonated under acidic conditions. In order to
investigate the fluorescence kinetics of ADB and ONOO–, the
time-dependent fluorescence signal of ADB and ONOO– was mea-
sured. As shown in Fig. 3d, before the addition of ONOO–, ADB itself
had a weak emission intensity. When 10 µM ONOO– was added,
the fluorescence intensity of ADB at 440 nm increased rapidly
and reached saturation with a very short time (about 20 s), the
time-dependent studies of the fluorescence changes indicated that
ADB could be used as a probe to detect ONOO–. In addition, the
photostability of ADB toward ONOO– was investigated. After 3 h
of ONOO– treatment, the fluorescence intensity of ADB was
observed to have no obvious change (Fig. S2), indicating that
ADB + ONOO– was stable and conductive to further study in the
biological system.
3.4. Proposed sensing mechanism
The sensing performance of the proposed method was com-
pared with that of other previously reported fluorescent probes
for ONOO–. One of the important merits of probe ADB was that it
Scheme 2. Solid state spectra of ADB under daily light (a) and UV lamp (c). Solid
state spectra of ADB with ONOO– under daily light (b) and UV lamp (d). (e)
possessed not only rapid sensing response, low detection limit
Proposed reaction mechanism of ADB with ONOO–.
and good water solubility, but also detecting ONOO– with AIE pro-
cess (Table S2). Next, to further prove the recognition mechanism
of ADB for detecting ONOO–. Boronate, as the responsive group
nate and finally obtained stable hydroxyl derivatives. And the reac-
tion product of ADB and ONOO– was confirmed by ESI-MS. As
of ADB, was used to react with ONOO–. For the nucleophilicity
and oxidizability, ONOO– caused a series of reactions with boro-
depicted in Fig. S3, the results showed that there was a peak at
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 261 (2021) 120044
quenching. These phenomena are consistent with the nature of
AIE.
3.5. Theoretical calculation
The theoretical investigations had been performed for AD to
understand the emission properties of ADB for detecting ONOO–.
We performed quantum chemical calculations by means of the
density functional theory (DFT) method at the B3LYP/6–31 + G
(d) level with Gaussian 09 programs to provide insights into the
structure optical properties of ADB for detecting ONOO–. As
depicted in Fig. 4 a, b, the calculated HOMO and LUMO energy
levels showed that the LUMO energy levels of AD increased and
the HOMO energy levels decreased compared with mOH, leading
to AD had bigger energy gaps (0.155 eV), suggesting that diethy-
lamino substituted positions could result in significant changes
in HOMO and LUMO energy levels, which in turn affected the emis-
sion wavelength. In addition, we also studied fluorescence
response of AD in different solvents and in different pH (Fig. 4 c,
d), the results showed that K-type emission peak of AD blue-
shifted and was consistent with changes of fluorescence spectra.
Accordingly, consulted with the published conclusion, such as
wang et al. used 2-(2-hydroxyphenyl) benzothiazole as a fluo-
rophore and introduced different electron-donating groups in the
meta positions of the hydroxyl group, and demonstrated that the
K-type emission peak blue-shifted as the electron donation was
enhanced.[34] Therefore, we also compared energy gaps AD and
p-NO2 (Fig. 5), as expected, AD had bigger energy gaps (0.155 eV)
than p-NO2 (0.118 eV), suggesting that K-type emission peak
blue-shifted of AD.
Fig. 4. Theoretical calculation of AD (a) and mOH (b). Fluorescence response of AD
in different solvents (c) and in different pH (d). Slit: 5/10.
299.1217 m/z, which corresponded to the product of ADB for
ONOO– (299.1218 m/z). We also did the UV and fluorescence
experiments, the red shift of absorption peak (Fig. S1a) and the
enhancement of fluorescence intensity of ADB with addition of
ONOO– (Fig. S4) was attributed to the fact that the reaction of
ADB with ONOO– promoted oxidative hydrolysis of the arly-
boronate group, which was in good agreement with the proposed
mechanism (Scheme 2e), and electronic transfer process of the
new recognition mechanism was shown Fig. S5. In addition, we
found that ADB showed brown under daily light (Scheme 2a) and
had no fluorescence in solid state under UV lamp (Scheme 2c).
However, the reaction product of ADB with ONOO– showed yellow
under daily light (Scheme 2b), and emitted strong light blue fluo-
rescence in solid state under UV lamp (Scheme 2d) that showed
a strong fluorescence emission peak at 437 nm (Fig. S6). In addi-
tion, the fluorescence emissions of AD in CH3CN/water mixed solu-
tions with different water content were examined (Fig. S7). The
results showed that the emission intensity of AD solutions gradu-
ally increased with increasing the water fraction from 0 to 70%.
Further increasing the water content resulted in fluorescence
3.6. Biological imaging in living cells
To test the application of ADB for bioimaging, the RAW 264.7
macrophage cells were chosen as criteria for evaluating the cyto-
toxicity of ADB. As shown in Fig. S8, the cell viability is still higher
than 80% while incubating RAW 264.7 macrophage cells with ADB
from 0 to 50 µM, which indicates the excellent biocompatibility
and ultra-low biological toxicity of ADB. Furthermore, fluorescence
images were collected on a confocal fluorescence microscope with
blue channels (Fig. 6). The macrophage cells only treated with ADB
exhibited almost no fluorescence under the excitation of 405 nm
laser, when introducing SIN-1 (an ONOO– donor) into the cells,
strong blue fluorescence was observed gradually and it demon-
strated effective detection in terms of the concentration of ONOO–,
Fig. 5. Theoretical calculation of AD (a) and p-NO2 (b).
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Declaration of Competing Interest
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Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.saa.2021.120044.
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