Two birds with one stone: The detection of nerve agents and AChE activity with an ICT-ESIPT-based fluorescence sensor

Article abstract of DOI:10.1016/j.jhazmat.2020.124811

Nerve agents are among the world's deadliest poisons, and the target enzyme is acetylcholinesterase (AChE). To better diagnosis nerve agent poisonings, a reliable diagnostic method for both nerve agents and AChE is desirable. Herein, we synthesized a series of fluorescent sensors for both real nerve agents and acetylcholinesterase activity detection. Among these sensors, HBQ-AE exhibited a fast response rate (within 10 s for nerve agent and 8 min for AChE), good sensitivity (the limit of detection is 6 nM and 0.2 U/mL) and a high off/on contrast. To the best of our knowledge, HBQ-AE is the first fluorescence sensor for nerve agents and AChE activity detection. The fluorescent change of HBQ-AE from nonfluorescence to blue fluorescence (nerve agent) or orange fluorescence (AChE) by excitation at 365 nm can be easily observed with the naked eye. HBQ-AE was successfully applied to image nerve agents and AChE activity in living cells. Moreover, HBQ-AE is the vital member to construct a test paper that can be employed to detect and diagnose chemical warfare agents.

Full text of DOI:10.1016/j.jhazmat.2020.124811

Journal of Hazardous Materials 410 (2021) 124811
Contents lists available at ScienceDirect
Journal of Hazardous Materials
journal homepage: www.elsevier.com/locate/jhazmat
Two birds with one stone: The detection of nerve agents and AChE activity
with an ICT-ESIPT-based fluorescence sensor
a
,
b
,
1
b
,
1
b
,
1
b
b
Wenqi Meng
, Zhipeng Pei , Yurun Wang , Mingxue Sun , Qingqiang Xu ,
b
a
b
,
*
a,*
Jinfeng Cen , Kai Guo , Kai Xiao , Zhenjiang Li
a
State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, China
b
Lab of Toxicology & Pharmacology, Faculty of Naval Medicine, Second Military Medical University, Shanghai, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Nerve agents are among the world’s deadliest poisons, and the target enzyme is acetylcholinesterase (AChE). To
better diagnosis nerve agent poisonings, a reliable diagnostic method for both nerve agents and AChE is desir-
able. Herein, we synthesized a series of fluorescent sensors for both real nerve agents and acetylcholinesterase
activity detection. Among these sensors, HBQ-AE exhibited a fast response rate (within 10 s for nerve agent and
Fluorescence sensor
Acetylcholinesterase
Nerve agent
Test paper
8
min for AChE), good sensitivity (the limit of detection is 6 nM and 0.2 U/mL) and a high off/on contrast. To the
best of our knowledge, HBQ-AE is the first fluorescence sensor for nerve agents and AChE activity detection. The
fluorescent change of HBQ-AE from nonfluorescence to blue fluorescence (nerve agent) or orange fluorescence
(
AChE) by excitation at 365 nm can be easily observed with the naked eye. HBQ-AE was successfully applied to
image nerve agents and AChE activity in living cells. Moreover, HBQ-AE is the vital member to construct a test
paper that can be employed to detect and diagnose chemical warfare agents.
even misleading, especially for inexperienced physicians. To diagnosis
nerve agent poisonings more scientifically, a reliable, accurate, and
convenient diagnostic method for nerve agents and their target enzyme
in human samples and in the environment is desirable.
1
. Introduction
Since the Syrian chemical weapon crisis (2013- present) (Dons,
013), the Malaysia VX poisonings (2017) (Hulse et al., 2019), and the
Current approaches to nerve agents and enzyme include electro-
chemistry, gas chromatography-mass spectrometry, Raman spectros-
copy and enzymatic bio-sensing technology (He et al., 2020; Khan et al.,
2
Salisbury Novichok poisonings (2018) (Butler, 2018), nerve agents have
been thrusted back into the spotlight. Nerve agents are a class of highly
toxic organophosphate compounds (Schwenk, 2018; Stone, 2018).
These agents can be absorbed various ways, such as through the skin,
ingestion, and inhalation. After resorption and distribution, their reac-
tive phosphate groups are able to irreversibly bind to the active site
serine of the pivotal enzyme acetylcholinesterase (AChE), which is
involved in hydrolysis of the neurotransmitter acetylcholine (ACh), to
inhibit the AChE activity (Amend et al., 2020; Kovarik et al., 2015). The
inability to hydrolyze ACh results in neurological problems, such as
salivation, convulsions, tremors, and even death (Amend et al., 2020).
Although nerve agent poisoning can be treated with a combination of
anticholinergic drugs and pyridinium oxime reactivators, treatment is
still difficult for many physicians (Henretig et al., 2019). The diagnosis
of nerve agent poisoning is traditionally based on the clinical appear-
ance (Ciottone, 2018). However, clinical signs might be inconclusive or
2
2
007; Lai et al., 2018; Liu and Lin, 2006; Seto et al., 2019; Wu et al.,
017). However, these methods often require large instruments,
complicated operation, and cumbersome sample pretreatment (Huo
et al., 2019; Jiang et al., 2011). Small-molecule fluorescent sensors have
been recognized as an effective tool for diagnosing toxic chemical poi-
sonings and many diseases because of the advantages of operational
simplicity, high sensitivity, low cost and a real-time response (Meng
et al., 2018a, 2018b, 2019a; Yadav et al., 2020; Zhang et al., 2019b,
2
019c). Due to the transient nature of nerve agents in human samples
and the nonexistence of AChE in environmental samples (Hemmert
et al., 2011; Schulze et al., 2016), fluorescent sensors are in demand to
detect nerve agents and AChE activity. To the best of our knowledge,
there is no fluorogenic sensor that can detect both a nerve agent itself
and AChE activity. However, some fluorogenic sensors that can detect
*
Corresponding authors.
E-mail addresses: kaixiaocn@163.com (K. Xiao), zjli@njtech.edu.cn (Z. Li).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.jhazmat.2020.124811
Received 2 October 2020; Received in revised form 20 November 2020; Accepted 6 December 2020
Available online 30 December 2020
0
304-3894/© 2020 Elsevier B.V. All rights reserved.

W. Meng et al.
Journal of Hazardous Materials 410 (2021) 124811
(
101 mg, 1.0 mmol), DMAP (61 mg, 0.5 mmol) and dimethylcarbamoyl
Nomenclature
chloride (54 mg, 0.5 mmol) in 10 mL of CH
The reaction mixture was cooled to room temperature and concentrated
under a vacuum. The residue was dissolved in H O (50 mL) and
extracted with DCM (2 × 10 mL). The organic fractions were combined
and dried over anhydrous MgSO , and the crude product was purified by
2 2
Cl was refluxed for 12 h.
AChE
ALP
Acetylcholinesterase
2
Alkaline phosphatase
BuChE
NMR
DCM
DCP
Butyrocholinesterase
4
column chromatography to obtain 69 mg white solid. 1H NMR (400
MHz, Chloroform-d) δ 8.00 (d, J = 8.7 Hz, 1 H), 7.85 (d, J = 9.3 Hz, 1 H),
7.33 (dd, J = 9.3, 2.8 Hz, 1 H), 7.11 (d, J = 8.7 Hz, 1 H), 6.84 (d, J = 2.8
Hz, 1 H), 3.17 (s, 3 H), 3.06 (s, 6 H), 3.03 (s, 3 H). 13C NMR (101 MHz,
Chloroform-d) δ 154.50, 154.20, 148.55, 139.88, 137.78, 129.02,
Nuclear magnetic resonance
Dichloromethane
Diethyl chlorophosphate
Dimethylaminopyridine
Ethylene diamine tetraacetic acid
Excited state intramolecular proton transfer
10-hydroxybenzo[h]quinoline
High resolution mass spectrometer
Intramolecular charge transfer
Limit of detection
DMAP
EDTA
ESIPT
HBQ
HRMS
ICT
128.38, 119.61, 116.03, 105.38, 40.80, 36.69. ESI-MS m/z: 260.1527
+
(Calcd for C14
H
18
N
3
O
2
(M+H) : 260.1399).
2.3.2. Synthesis of compound QN-pAE
LOD
Compound 9 was derived from diethyl ethoxymethylenemalonate
according to Suzuki et al. (2007). A mixture of compound 9 (94 mg, 0.5
mmol) and TEA (101 mg, 1.0 mmol), DMAP (61 mg, 0.5 mmol) and
PPE
Porcine pancreatic elastase
Quinoline
QN
TEA
Triethylamine
dimethylcarbamoyl chloride (54 mg, 0.5 mmol) in 10 mL of CH
refluxed for 12 h. The reaction mixture was cooled to room temperature
and concentrated under a vacuum. The residue was dissolved in H
2 2
Cl was
2
O
(
50 mL) and extracted with DCM (2 × 10 mL). The organic fractions
only nerve agents or AChE activity have been developed. Yoon’s and
Churchill’s group reported a series of nerve agent sensors and reviewed
the development of chemosensors for nerve agents (mimic) (Chen et al.,
4
were combined and dried over anhydrous MgSO , and the crude product
1
was purified by column chromatography to obtain 57 mg white solid. H
NMR (400 MHz, Chloroform-d) δ 8.58 (d, J = 4.9 Hz, 1 H), 7.94 (d, J =
2
2
018; Jang et al., 2015; Kim et al., 2017; Zeng et al., 2019; Zhou et al.,
016). And Tang and Guo groups also recently reported two high
9
6
.3 Hz, 1 H), 7.35 (dd, J = 9.3, 2.9 Hz, 1 H), 7.23 (d, J = 4.9 Hz, 1 H),
13
.88 (d, J = 2.9 Hz, 1 H), 3.25 (s, 3 H), 3.09 (s, 3 H), 3.07 (s, 6 H).
C
selectivity fluorescent sensors for AChE activity (Ma et al., 2020; Wang
et al., 2019b). These progresses inspired us to develop dual-response
fluorescent sensors for the detection of nerve agents and acetylcholin-
esterase to diagnose nerve agent poisonings.
NMR (101 MHz, Chloroform-d) δ 153.46, 153.04, 148.64, 146.37,
1
44.23, 130.15, 123.96, 119.65, 112.51, 98.07, 40.61, 36.94, 36.66.
+
ESI-MS m/z: 260.1403 (Calcd for C14
H
18
3
N O
2
(M+H) : 260.1399).
Herein, we present the design and synthesis of a series of fluorescent
sensors that detect real nerve agents and AChE activity. The most
advanced member of this series is HBQ-AE, which can show nerve agent
concentrations and AChE activity in different emissions with one exci-
tation wavelength, with detection limits as low as 6 nM for nerve agents
and 0.2 U/mL for AChE activity. HBQ-AE was also applied to visualize
nerve agent concentration and AChE activity in live cells. Furthermore,
we employed HBQ-AE to diagnose nerve agent poisonings by detecting
AChE activity in whole blood. Finally, HBQ-AE was employed as the vital
member of a test paper to detect and diagnose chemical warfare agents.
2
.3.3. Synthesis of compound HBQ-AE
A mixture of compound 10 (98 mg, 0.5 mmol) and TEA (101 mg, 1.0
mmol), DMAP (61 mg, 0.5 mmol) and dimethylcarbamoyl chloride (54
mg, 0.5 mmol) in 10 mL of CH Cl was refluxed for 12 h. The reaction
mixture was cooled to room temperature and concentrated under a
vacuum. The residue was dissolved in H O (50 mL) and extracted with
DCM (2 × 10 mL). The organic fractions were combined and dried over
2
2
2
4
anhydrous MgSO , and the crude product was purified by column
chromatography to obtain 76 mg white solid. 1H NMR (400 MHz,
Chloroform-d) δ 8.88 (dd, J = 4.3, 1.9 Hz, 1 H), 8.12 (dd, J = 8.1, 1.9 Hz,
1
3
1
1
2
H), 7.84 – 7.77 (m, 2 H), 7.66 (t, J = 7.9 Hz, 2 H), 7.49 – 7.41 (m, 2 H),
.38 (s, 3 H), 3.11 (s, 3 H). 13C NMR (101 MHz, Chloroform-d) δ 156.26,
49.72, 147.82, 146.23, 136.03, 135.52, 128.11, 128.09, 127.29,
2
. Experimental
2
.1. Safety statement
26.20, 126.12, 124.15, 122.86, 121.35, 37.00, 36.95. ESI-MS m/z:
+
67.1722 (Calcd for C16
H
15
N
2
O
2
(M+H) : 267.1134).
Soman and sarin are highly toxic chemical warfare agents. A rec-
ommended operating procedure must be followed during the use of
nerve agents. Protection equipment should be adopted, especially a full-
face mask.
2
.3.4. Synthesis of compound QN-oME
Compound 5 was derived from ethoxyethane according to Janiak
et al. (1999). A mixture of the compound 5 (94 mg, 0.5 mmol) and TEA
101 mg, 1.0 mmol), DMAP (61 mg, 0.5 mmol) and acetyl chloride (39
mg, 0.5 mmol) in 10 mL of CH Cl was refluxed for 16 h. The reaction
(
2
.2. Materials
2
2
mixture was cooled to room temperature and concentrated under a
Soman, sarin, sulfur mustard and potassium cyanide were provided
vacuum. The residue was dissolved in H O (50 mL) and extracted with
2
by the Institute of Chemical Defense (China). Unless otherwise illus-
trated, all chemical reagents were analytical reagent grade and used
without further purification. An NMR was obtained using a Bruker 400/
CH Cl (2 × 10 mL). The organic fractions were combined and dried
2
2
over anhydrous MgSO , and the crude product was purified by column
4
1
chromatography to obtain 78 mg white solid. H NMR (400 MHz,
5
00 spectrometer. High-resolution mass spectra were obtained on an
Chloroform-d) δ 8.04 (d, J = 8.7 Hz, 1 H), 8.02 (d, J = 9.3 Hz, 1 H), 7.86
(dd, J = 9.3, 2.8 Hz, 1 H), 7.83 (d, J = 8.7 Hz, 1 H), 6.84 (d, J = 2.8 Hz, 1
H), 3.06 (s, 6 H), 2.38 (s, 3 H). 13C NMR (101 MHz, Chloroform-d) δ
Agilent 6520 Q-TOF LC/MS Spectrometer. The fluorescence spectra
were obtained on a Hitachi F-2710 fluorescence spectrophotometer.
1
69.56, 153.39, 148.77, 141.01, 137.97, 129.03, 128.66, 119.83,
2
2
.3. Synthesis and characterization of compounds
115.58, 105.18, 40.73, 21.32. ESI-MS m/z: 231.1126 (Calcd for
+
C
13
H
15
N
2
O
2
(M+H) : 231.1134).
.3.1. Synthesis of compound QN-oAE
Compound 5 was derived from ethoxyethane according to Janiak
2.3.5. Synthesis of compound QN-pME
et al. (1999). A mixture of the compound 5 (94 mg, 0.5 mmol) and TEA
A mixture of compound 9 (94 mg, 0.5 mmol) and TEA (101 mg, 1.0
2

W. Meng et al.
Journal of Hazardous Materials 410 (2021) 124811
mmol), DMAP (61 mg, 0.5 mmol) and acetyl chloride (39 mg, 0.5 mmol)
primers based on the gene sequence of AChE and used in amplification.
The product was cloned into the simple vector to generate recombinant
plasmid, which was identified by enzyme digestion and sequencing.
The cells were then transfected with the recombinant plasmid. After
treatment for 24 h, we detected the relative expression of AChE in
cells through PCR experiment to ensure that the purpose of over-
expression of AChE was successfully achieved. To overexpress or
in 10 mL of CH
cooled to room temperature and concentrated under a vacuum. The
residue was dissolved in H
O (50 mL) and extracted with DCM (2 × 10
mL). The organic fractions were combined and dried over anhydrous
MgSO , and the crude product was purified by column chromatography
to obtain 86 mg white solid. H NMR (400 MHz, Methanol-d
J = 7.1 Hz, 1 H), 7.49 (d, J = 8.8 Hz, 1 H), 7.37 (d, J = 9.0 Hz, 2 H), 6.28
2 2
Cl was refluxed for 16 h. The reaction mixture was
2
4
1
4
) δ 7.84 (d,
knockdown AChE gene, PC12 cells upon reaching 60–70% confluence
1
3
′
(
d, J = 7.0 Hz, 1 H), 3.03 (s, 6 H), 1.99 (s, 2 H). C NMR (101 MHz,
were
infected
with
the
AChE
plasmid
(Forward:
5 -
′
Methanol-d
4
) δ 178.44, 148.12, 137.87, 132.40, 126.49, 120.50,
18.97, 106.70, 103.49, 39.57, 19.44. ESI-MS m/z: 231.1143 (Calcd for
TAGAGCTAGCGAATTCATGAGGCCTCCCTGGTATCCC-3 , Reversed:
′
′
1
5 - TTGCGGCCGCGGATCCCAGGTCTGAGCAGCGCTCC-3 ) or siRNA
′ ′
specific to AChE (sense: 5 -GCUCAGCGACUUAUGAAAUTT-3 , anti-
+
C
13
H
15
2
N O
2
(M+H) : 231.1134).
′
′
sense: 5 - AUUUCAUAAGUCGCUGAGCAA-3 ) using lipofectamine
3000 (Invitrogen, Life Technologies, Carlsbad, CA, USA) as previously
described, respectively. After treatment for 24 h, cells were used in
further experiments. Cells were harvested and transferred to a cover
glass, where the cells grew to 80% confluence. HBQ-AE was added to
the cell media and incubated for 15 min. The cells were then washed
with PBS (1 mL×3 times), and culture media were added. Some other
agents were added to the cell media and incubated for different
experimental objectives. Confocal fluorescence imaging was performed
using an Olympus FV1000 confocal laser scanning microscope with a
60 × oil immersion objective lens. The excitation wavelength was 405
nm. Emission was collected at a wavelength of 420–480 nm/520–620
nm. The images were further processed with Olympus software.
2
.3.6. Synthesis of compound HBQ-ME
A mixture of compound 10 (98 mg, 0.5 mmol) and TEA (101 mg, 1.0
mmol), DMAP (61 mg, 0.5 mmol) and acetyl chloride (39 mg, 0.5 mmol)
in 10 mL of CH Cl was refluxed for 16 h. The reaction mixture was
cooled to room temperature and concentrated under a vacuum. The
residue was dissolved in H
O (50 mL) and extracted with DCM (2 × 10
mL). The organic fractions were combined and dried over anhydrous
MgSO , and the crude product was purified by column chromatography
to obtain 82 mg white solid. H NMR (400 MHz, Chloroform-d) δ 8.97
2
2
2
4
1
(
dd, J = 4.3, 1.8 Hz, 1 H), 8.14 (dd, J = 8.0, 1.8 Hz, 1 H), 7.83 (dd, J =
8
7
.3, 6.8 Hz, 2 H), 7.72 – 7.65 (m, 2 H), 7.50 (dd, J = 8.0, 4.3 Hz, 1 H),
13
.41 (dd, J = 7.7, 1.3 Hz, 1 H), 2.60 (s, 3 H). C NMR (101 MHz,
Chloroform-d) δ 170.02, 147.66, 147.03, 144.56, 135.00, 134.69,
1
2
2
27.04 (d, J = 7.3 Hz), 126.22, 125.66, 125.35, 122.26, 121.13, 120.51,
2.7. Flow cytometric analysis
+
0.70. ESI-MS m/z: 238.0861 (Calcd for
C
15
H12NO
2
(M+H) :
38.0868).
Cells were treated with cisplatin at 50
μ
g/mL for 6 h and then
digested by EDTA-free trypsin. After being washed in precold PBS, the
cells were resuspended with 100 µL binding buffer. Then, 5 µL annexin
V-FITC and 5 µL PI working solution were added and incubated for 15
min at room temperature in the dark (Dojindo Co., Kumamoto, Japan).
Cellular apoptosis in different groups was then analyzed by flow cy-
tometer (Beckman Coulter, Miami, USA).
2
.4. Fluorometric measurements
As we previously mentioned (Meng et al., 2018a, 2018b), All fluo-
rescence measurements were performed at room temperature on a
Hitachi F-2710 fluorescence spectrophotometer. The probe solution
(
3
CH CN) was added to a quartz cuvette. The fluorescence intensity was
recorded (λex = 365 nm) with the slit widths set at 10 nm for excitation
2.8. Western blotting of AChE
and emission. The emission spectra ranged from 390 to 600 nm,
4
00–650 nm, and 500–700 nm.
As we previously mentioned (Zhang et al., 2019a), cells were washed
with precold PBS and then lysed in RIPA buffer supplemented with
protease inhibitors. The protein contents were quantified with BCA
protein assay kits (Beyotime Biotechnology, Nanjing, China). For west-
2
.5. Molecular docking
◦
A Surflex molecular docking module in Sybyl X-2.1 (Sybyl X-2.1 is
ern blot analysis, the extracted protein was boiled for 10 min at 100 C in
available from Tripos Associates Inc., S Hanley Rd., St. Louis, MO
31444, USA) was used for investigating the possible binding interac-
a 5 × loading buffer, separated on 10% SDS-PAGE, and then transferred
onto polyvinylidene difluoride membranes (PVDF; Millipore, Whatman)
at 300 mA for 90 min. Five percent skim milk powder was used for
6
tion between human acetylcholinesterase and the compound HBQ-AE.
The crystal structure of human acetylcholinesterase complex with Ter-
ritrem B (PDB ID: 4m0f) was obtained from the RCSB Protein Data Bank.
The 3D structure of the compound HBQ-AE was modeled via the sketch
module in Sybyl-X2.1 and charged in Gasteiger–Huckel. Then, com-
pound HBQ-AE was minimized using the standard Tripos force field
until an energy gradient convergence criterion of 0.001 kcal/(mol⋅Å)
was achieved as described in previous studies (Gao et al., 2020; Liang
et al., 2018). The location of ligand Territrem B was defined as the
binding site of the compound HBQ-AE, and all water molecules were
removed. The missing hydrogen atoms of acetylcholinesterase were
added using the biopolymer module during protein preparation, and the
docking parameters were kept as default values. The binding affinity of
the compound HBQ-AE to human acetylcholinesterase was evaluated
with the docking score.
◦
blocking the membranes in a washing buffer for 1 h at 25 C. The
blocked membranes were incubated with primary antibodies overnight
◦
at 4 C. After rinsing, each membrane was incubated with secondary
antibodies for 1 h at room temperature, followed by detection with an
enhanced chemiluminescence system (ECL Western Blotting System;
Amersham Bioscence). Protein bands were quantified and analyzed
using ImageJ version 1.34 s software.
2.9. Animals and administration
Eight-week-old male C57BL6/J mice were purchased from the ani-
mal center of Second Military University. All mice were housed in a
temperature and light-controlled environment (22 ± 2 ◦C; 12 h light/
dark cycle). In addition, the animals were fed a standard laboratory diet
and pure water. The mice were adapted for a week prior to the experi-
ment. Nine mice were randomized into three groups (n = 6 each): the
control group, intraperitoneal injection of saline (200 µL); the soman
group, intraperitoneal injection of soman (5 µg/kg); and the cyanide
group, intraperitoneal injection of potassium cyanide (700 µg/kg). Mice
were anesthetized with ketamine/xylazine after 30 min, and blood was
2
.6. Cell culture and confocal imaging of cells
As previously mentioned (Feng et al., 2019; Qin et al., 2020), PC12
cells were grown in high-glucose DMEM supplemented with 10% fetal
bovine serum and penicillin/streptomycin. We designed specific
3

W. Meng et al.
Journal of Hazardous Materials 410 (2021) 124811
Fig. 1. Design strategy of sensors and fluorescence signal changes.
drawn with a heparinized (~50 USP/mL) syringe from the inferior vena
cava of male C57BL6/J mice.
mL distilled water. 0.1 mL mixed solution was used for detection. 0.5 mL
substrate buffer and 0.5 mL chromogenic solution were added to the
detection solution, mixed evenly, and reacted at 37 ℃ for 6 min. Then,
0
.03 mL inhibitor buffer and 0.1 mL transparent solution were added.
2
2
.10. Determination of AChE in the whole blood
The absorbance at 412 nm was determined by a microplate reader after
standing at room temperature for 15 min. AChE activity was calculated
by comparing the standard solution and control solution.
.10.1. With fluorescence sensor HBQ-AE
Measurements of the AChE activity were further modified based on
our previously described method (Meng et al., 2017, 2019b; Qian et al.,
012). Anticoagulated blood was diluted with distilled water in the ratio
2
.11. Ethics statement
2
of 1:99. A 100 µL diluted sample was added into 860 µL PBS buffer, and
different concentrations of AChE solution (20 µL) were spiked into the
sample as an internal standard. HBQ-AE was then added. Emission
spectra (λex = 365 nm, emission at 560 nm) were collected after the
Animal experiments were performed according to the regulations
and guidelines approved by the Animals Research Committee at Second
Military Medical University (SMMU, License No. 2011023). The animals
received humane care throughout all the procedures in accordance with
the Guide for the Care and Use of Laboratory Animals, published by the
US National Institutes of Health (Publication No. 85-23, revised 1996).
◦
mixture was incubated at 37 C for 10 min. The data represent the
average of three independent experiments.
2
.10.2. With a commercial detection kit
Working solutions were prepared according to the kit (Nanjing
3. Results and discussion
Jiancheng Bioengineering Institute Biotechnology, Nanjing, China) in-
structions. Anticoagulated blood was diluted with distilled water in the
ratio of 1:99, such that 0.1 mL of anticoagulated blood was added to 10
The reaction time is the most crucial factor for nerve agent detection
because of the quick killing effect. Until now, among the various
Scheme 1. Synthesis and structure of probes. Conditions: (a) COCl
TEA, DMAP, CH Cl , reflux, 12 h; (d) toluene, reflux, 1 h, followed by diphenylether, reflux, 1 h; (e) 2 N-NaOH, reflux, 1 h, followed by diphenylether, reflux, 1 h; (f)
TEA, DMAP, CH Cl , reflux, 16 h.
2
, 20% in toluene, rt, 2 h; (b) TEA, toluene, 100–110 ℃, 1 h, followed by H
2 4
SO , 24 h, 50 ℃; (c)
2
2
2
2
4

W. Meng et al.
Journal of Hazardous Materials 410 (2021) 124811
Fig. 2. Fluorescence spectral changes of sensors upon addition of various concentrations of soman (0–80
μ
M)/AChE (0–20 U/mL). All the data were collected using a
sensor concentration of 20 M at room temperature for 1 min/10 min. The excitation wavelength was 365 nm.
μ
receptor units for nerve agent detection, pyridine showed a relatively
fast rate with nerve agents (Cai et al., 2017; Kim et al., 2017). Tang and
Guo reported that dimethyl carbamate might be the suitable recognition
group for selective reactivity with AChE, and it was inspired by AChE
inhibitors. Thus, pyridine and dimethyl carbamate were chosen as the
recognition groups for detecting nerve agents and AChE (Fig. 1). To
combine two recognition groups in one molecule, we employed some
suitable fluorophores that had both pyridine and oxhydryl groups.
Recently, 10-hydroxybenzo[h]quinoline (HBQ), an excited-state intra-
molecular proton transfer (ESIPT) fluorophore, attracted attention
With these compounds in hand, we examined the detection perfor-
mance for nerve agents and AChE. HBQ-AE and HBQ-ME showed almost
no fluorescence; however, upon reacting with different concentration of
soman, HBQ-AE and HBQ-ME showed a significant increase in fluores-
cence signals at 450 nm and 441 nm, respectively (Fig. 2a). QN-pAE,
QN-oAE, QN-pME, and QN-pME have rather strong fluorescence emis-
sions at 454 nm, 458 nm, 445 nm and 446 nm, respectively. Upon
treatment of these sensors with a cascade of soman, fluorescence in-
tensity gradually decreased (Fig. 2a). These sensors could also react with
Sarin and diethyl chlorophosphate (DCP, a commonly used nerve agent
mimic) accompanied by similar significant fluorescence signals of
soman (Fig. S1), which suggested that these sensors could react with
nerve agents. Next, the responses of sensors elicited by AChE were also
explored by using fluorescence spectroscopy. As shown in Fig. 2(b) and
S2, all sensors showed significant fluorescence changes after treatment
with AChE. Both AChE and BuChE (butyrocholinesterase) are cholin-
esterase, and BuChE also showed similar catalytic performance with
AChE (Ma et al., 2020; Wang et al., 2019b). To investigate the selectivity
of all sensors towards AChE, all sensors were treated with BuChE. As
shown in Fig. S2, HBQ-AE, QN-pAE and QN-oAE could react with AChE,
(
Wang et al., 2019a; Wu et al., 2019). In addition, Quinolines are also
commonly used fluorescence dyes (Dias et al., 2018; Jun et al., 2018).
Thus, we selected HBQ and two quinolines as fluorophores, meanwhile
pyridine and dimethyl carbamate were chosen as the recognition group
for nerve agents and AChE, respectively. We synthesized all sensors by
using an esterification reaction to convert commercially available fluo-
rophores into a dual-response fluorescent sensor to detect nerve agents
1
and AChE (Scheme 1). All of these sensors were characterized by
H
1
3
NMR, C NMR and HRMS, as presented in the Supporting Information
Fig. S9–S26).
(
5

W. Meng et al.
Journal of Hazardous Materials 410 (2021) 124811
Fig. 3. The selectivity of HBQ-AE for nerve
agents and AChE. (a) 1. Blank; 2. Soman
(
(
(
80
μ
M); 3. Sulfur mustard (200
μ
M); 4. KCN
M); 6. Lewisite agent
M); 7. NaOH (200 M); 8. Phosphoryl
M); 9. Iodoethane (200 M);
M). The excitation
200
200
μ
μ
M); 5. NaCN (200
μ
μ
trichloride (200
μ
μ
1
0. Ethyl bromide (200
μ
wavelength was 365 nm, the fluorescence in-
tensity of emission wavelength was record at
4
50 nm. (b) 1. Blank; 2. AChE (20 U/mL); 3.
ALP (40 U/mL); 4. PPE (40 U/mL); 5. Trypsin
40 U/mL); 6. Chymostrypsin (40 U/mL); 7.
(
Lysozyme (40 U/mL); 8. Phosphdipase (40 U/
mL); 9. Carboxypeptidase (40 U/mL); 10. Car-
boxylesterase (40 U/mL). The excitation wave-
length was 365 nm, the fluorescence intensity
of emission wavelength was record at 560 nm.
Data are presented as the mean ± SD (n = 3).
Fig. 4. The possible detection mechanism of nerve agents and AChE using HBQ-AE. (For interpretation of the references to colour in this figure, the reader is referred
to the web version of this article).
while they did not react with BuChE. These results suggested that
HBQ-AE, QN-pAE and QN-oAE could react with AChE selectivity. Then
we observed the reaction time of all sensors towards soman and AChE.
As shown in Fig. S3a, all sensors finished reacting with soman in short
time (10–60 s), which HBQ-AE could finish reacting in 10 s. Meanwhile,
the fluorescence intensity in the reaction of HBQ-AE to AChE reached
the maximum value within 8 min (Fig. S3b). We also explored the linear
detection range and detection limit of sensors. The linear detection
interference with nerve agents and AChE detection in the coexistence of
nerve agents/AChE and various analytes. Thus, these results suggested
that HBQ-AE can be used for the selective detection of nerve agents and
AChE.
Then, we further explored the reaction mechanism of HBQ-AE. The
reaction mechanisms of HBQ-AE with nerve agent and AChE was pro-
posed in Fig. 4 according to Song and Tang’ report (Cai et al., 2017;
Wang et al., 2019b). AChE-triggered cleavage of the carbamyl group on
HBQ-AE, which induced the formation of HBQ. The generated HBQ
displayed strong orange fluorescence. For nerve agent, the pryridine
unit of HBQ-AE could be catalytic hydrolysis to form the protonated
HBQ-AE, which often exhibits a stronger ICT process. The products of
HBQ-AE with nerve agents/AChE were characterized by HRMS (please
refer to the Supplementary Information for details), demonstrating that
the reactions of HBQ-AE with nerve agents/AChE proceeded as designed
in Fig. 4.
range of sensors for soman was 0–40
and HBQ-ME), 0–30 M (QN-oME), and 0–15
/κ, the detection limit of sensors for soman was 2.80
QN-pAE), 0.98 M (QN-pME), 3.5 M (QN-pME), 2.4 M (QN-oME),
nM (HBQ-AE), 0.03 M (HBQ-ME). In addition, the limits of detection
μ
M (QN-pAE, QN-pME, HBQ-AE,
μ
μ
M (QN-oAE). According
to LOD= 3
σ
μ
M
(
μ
μ
μ
6
μ
of QN-oAE, HBQ-AE, and QN-pAE to AChE were 1.6 U/mL, 0.2 U/mL
and 0.9 U/mL, respectively. We also summarized the fluorescent probes
for nerve agents and AChE activity (Table S1). Taken together, among
these sensors HBQ-AE displayed the best performance in terms of the
aspects of linear detection range, response time, and detection limit.
To investigate the selectivity of HBQ-AE towards nerve agents and
AChE, HBQ-AE was treated with various analytes, including chemical
warfare agents, common hydrolases and other interferent substances.
HBQ-AE showed high selectivity for nerve agents over interferent sub-
stances and other chemical warfare agents, including Sulfur mustard,
KCN, NaCN, Lewisite agent, NaOH, Phosphoryl trichloride, Iodoethane,
Ethyl bromide (Fig. 3a). And HBQ-AE was highly sensitive to AChE
compared with many tested hydrolases, including Alkaline phosphatase
To further understand the sensing mechanism of HBQ-AE, we per-
formed theoretical and computational experiments. Per the results of
scan of total energy of HBQ (enol) and HBQ (keto), there was no energy
barrier between HBQ (enol) and HBQ (keto), suggesting that HBQ (enol)
was not observed in the excitation process (Fig. S4). Thus, we could
observe only one fluorescence emission of HBQ instead of the double
fluorescence emissions. Then, we calculated the electronic transition
energies for HBQ-AE and its reaction products (Fig. 5a). HBQ is an ESIPT
fluorophore, and the ESIPT process of HBQ is blocked using an ester
protecting group. The group can then be selectively removed by AChE,
resulting in a turn-on fluorescence-based sensor suitable for the detec-
tion of AChE. After reacting with nerve agents, electron promotion from
the highest occupied molecular orbital to the lowest unoccupied
(
ALP), porcine pancreatic elastase (PPE), Trypsin, Chymostrypsin,
Lysozyme, Phosphdipase, Carboxypeptidase, Carboxylesterase (Fig. 3a).
Additionally, competitive experiments also revealed hardly any
6

W. Meng et al.
Journal of Hazardous Materials 410 (2021) 124811
Fig. 5. Theoretical and computational experiments of HBQ-AE. (a) Plots of the HOMO and LUMO of the ground state for HBQ-AE and its reaction products. (b)
Ensemble docking studies of AChE and HBQ-AE.
Fig. 6. (a) The induction of apoptosis in PC12 cells was determined by Annexin V/PI flow cytometry following treatment with or without cisplatin (50
μ
g/mL) for
6
h. (b) The protein levels of AChE were examined via western blotting. (c) Fluorescence images of PC12 cells for AChE activity. (d) Relative average fluorescence
intensities of all cells. Data are presented as the mean ± SD (n = 3). ** p < 0.01 vs. control. (For interpretation of the references to colour in this figure, the reader is
referred to the web version of this article).
7

W. Meng et al.
Journal of Hazardous Materials 410 (2021) 124811
molecular orbital is accompanied by a charge transfer from the donor
moiety to the acceptor moiety, which suggests the sensing mechanism of
HBQ-AE could be classified as ESIPT/ICT. After promotion, HBQ-AE was
docked to AChE (PDB 40 mf). A molecular docking study showed that
HBQ-AE fit well into the binding site of human acetylcholinesterase
showed much stronger fluorescence than untreated cells (Fig. 6c). These
results revealed the capability of HBQ-AE to recognize AChE activity in
living cells.
We further assessed the potential utility of HBQ-AE to monitor both
AChE and nerve agents in living cells. In the control group, cells loaded
(
Fig. 5b). It can be seen that HBQ-AE formed three hydrogen bonds, with
with HBQ-AE (20 M) exhibited almost no fluorescence in the blue
μ
side chains of residues Tyr72 and Asp74 and a main chain of Thr75.
Moreover, HBQ-AE was accommodated into a well-defined hydrophobic
pocket, which consisted of residues Tyr72, Leu76, Tyr124, Trp286,
Phe297 and Tyr341. It is noteworthy that the aromatic ring group of
channel (Fig. 7a and S7), but we could observe the fluorescence in the
orange channel (Fig. 7a). In the low-dose nerve agent group, after being
pretreated with soman (10
μ
M), PC12 cells loaded with HBQ-AE (20 M)
μ
exhibited obvious fluorescence in the blue channel (Fig. 7b), and almost
no orange fluorescence could be imaged (Fig. 7b). The data indicated
that the orange fluorescence change of HBQ-AE in cells arises from AchE.
HBQ-AE had a strong
π
-π stacking interaction with the residue Trp286.
Thus, the binding of HBQ-AE to human acetylcholinesterase was almost
dominated by a hydrogen bond and hydrophobic interactions.
In the high-dose nerve agent group, after addition of soman (20
μ
M) to
To explore the ability of HBQ-AE to detect AChE activity in living
cells, we measured the activity and expression of AChE. First, the
cytotoxicity of HBQ-AE was measured with CCK8 assays. As shown in
Fig. S5, the sensor and reaction product showed almost no effect on cell
viability, which suggested that cells were variable after incubation with
HBQ-AE for 24 h. Next, we used HBQ-AE to image AChE activity in
living PC12 cells. The time for HBQ-AE fluorescence increase was about
cells, PC12 cells loaded with HBQ-AE (20 M) showed significant blue
μ
fluorescence (Fig. 7c), and there was no fluorescence emission intensity
in orange channel (Fig. 7c). These results suggested that HBQ-AE is
capable of imaging both AChE and nerve agents in living cells.
Then, we applied HBQ-AE to the measurement of AChE activity in
mouse blood. Accordingly, the measurement of AChE activity in mouse
blood was conducted based on our previous method (Meng et al., 2017,
2019b; Qian et al., 2012). As shown in Fig. S8(a), the AChE activity in
mouse blood was 18.20 ± 1.47 U/mL. The validity of our present
method was verified using an acetylcholinesterase activity assay kit. The
values obtained by HBQ-AE were similar to those obtained by com-
mercial kits (Fig. S8a), suggesting that HBQ-AE is suitable for deter-
mining AChE activity in mouse blood. HBQ-AE was then applied to
detect the levels of AChE in the blood of mice treated with soman and
cyanide. As shown in Table 1, the AChE activity of the soman group was
significantly lower compared to that of the control mice. In addition, the
AChE activity of the cyanide group was almost equal to the activity in
the control mice. These results suggested that HBQ-AE could detect
AChE activity in mouse blood.
1
0 min (Fig. S6). It has been reported that apoptosis could increase the
AChE expression (Zhang and Greenberg, 2012; Zhang et al., 2002). PC12
cells were pretreated with cisplatin for 6 h. Flow cytometry was
employed to confirm the apoptosis caused by cisplatin (Fig. 6a). We also
detected the expression of AChE by western blot (Fig. 6b). These results
indicated that treatment with cisplatin could increase the expression of
AChE. As shown in Fig. 6(c), the images of PC12 cells treated with
cisplatin showed much stronger fluorescence than the untreated cells.
Next, we constructed cells with AChE knockdown and overexpression
(
Fig. 6b). As shown in Fig. 6(c), PC12 cells that were knockdown AChE
showed weaker fluorescence in orange channel than cells from the
control group. In contrast, the fluorescence of AChE overexpression cells
Fig. 7. Fluorescence images of PC12 cells for nerve agents and AChE activity. (a) Cells were incubated with HBQ-AE alone for 10 min (b) Cells were pretreated with
soman (10 M) for 10 min (c) Cells were pretreated with soman (20
μ
μM) for 10 min (d) Relative fluorescence intensities of the blue and orange channels in group a–c.
Data are presented as the mean ± SD (n = 3). (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article).
8

W. Meng et al.
Journal of Hazardous Materials 410 (2021) 124811
Table 1
poisoning detection and diagnosis. This research not only offers a
practical tool for nerve agent/AChE-related studies, but it also provides
insights into devising an effective strategy for the diagnosis of toxic
compound poisoning by detecting both the intact compound itself and
the target enzyme.
Measurement of AChE activity in mouse blood using HBQ-AE (n = 6).
Samples
Control (U/mL)
Soman (U/mL)
Cyanide (U/mL)
1
17.29
8.78
18.46
2
18.68
6.22
15.63
3
19.55
4.56
16.59
4
21.83
8.73
19.54
CRediT authorship contribution statement
5
19.52
4.92
18.77
6
14.83
6.88
20.82
Wenqi Meng: Conceptualization, Methodology, Investigation,
Formal analysis, Writing - original draft, Resources, Funding acquisition,
Writing - review & editing. Zhipeng Pei: Project administration, Writing
Average
18.62 ± 2.37
6.68 ± 1.81
18.30 ± 1.90
Inspired by the highly desirable features of HBQ-AE, HBQ-AE was
used as a vital component to construct a portable test paper for chemical
warfare agent detection and diagnosis. As shown in Fig. 8(a), the test
paper contained HBQ-AE, OXSM-1 and two miscellaneous reagents.
-
review & editing. Yurun Wang: Project administration, Validation.
Mingxue Sun: Data curation, Investigation. Qingqiang Xu: Data
curation, Investigation. Jinfeng Cen: Data curation, Investigation. Kai
Guo: Data curation, Investigation. Kai Xiao: Funding acquisition,
Writing - review & editing. Zhenjiang Li: Conceptualization, Method-
ology, Writing - review & editing.
After exposure of the test paper to soman (10 M) for 2 s, the bar 4 with
μ
HBQ-AE showed a strong blue fluorescence (Fig. 8b). Bar 1 of the test
paper showed significant color changes from brown to orange. We can
also detect sulfur mustard (10
μ
M) and cyanide (10 M) by using the test
μ
Declaration of Competing Interest
paper (Fig. 8c and d). Furthermore, exposure of the test paper to mouse
diluted blood (with distilled water in the ratio of 1:4) of the control
group and cyanide group promoted a change in the fluorescence signal
from no fluorescence to obvious orange fluorescence (Fig. 8e and g). In
contrast, mouse blood of the soman group did not induce significant
orange fluorescence. These results indicate that the strategy we devised
has led to the development of a simple test paper for rapid detection of
nerve agents and AChE activity.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by the National Natural Science Foundation
of China (81801869 and 81671858), Shanghai Municipal Health
Commission-Outstanding Youth Foundation of Public Health (GWV-
10.2-YQ18) and the Scientific Research Foundation of Second Military
Medical University (19WLMS-08 and 2017JS25).
4
. Conclusions
In summary, we have developed the first reported fluorescent sensor
for selective detection of real nerve agents and AChE activity, which
would provide some new insights in molecular design of fluorescence
sensors. The sensor HBQ-AE, which uses ESIPT/ICT, reacts rapidly and
with high selectivity and sensitivity. Using HBQ-AE, nerve agents and
AChE can be imaged in living cells. Furthermore, HBQ-AE could di-
agnose nerve agent poisoning by detecting AChE activity in mouse
blood. Finally, based on HBQ-AE and some other sensors, a practical test
paper was developed in this study. The test paper might be an important
supplementary method to be employed for the chemical warfare agent
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.jhazmat.2020.124811.
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