A FRET-based ratiometric fluorescent probe for highly selective detection of cysteine based on a coumarin-rhodol derivative

Article abstract of DOI:10.1039/c9nj03375k

Cysteine, as an important amino acid in the human body, plays a vital role in people's normal life activities. In this paper, a ratiometric fluorescent probe for detecting cysteine was designed and synthesized based on the fluorescence resonance energy transfer (FRET) process. In this FRET system, a coumarin derivative was used as the energy donor, a rhodol fluorophore was chosen as the energy receptor, and an acrylate group was utilized as a cysteine recognition unit. In the absence of cysteine, the rhodol receptor was in the non-fluorescent lactone state and the FRET process was inhibited. Upon addition of cysteine, the closed spirolactone form was converted to a conjugated fluorescent xanthene form to induce the occurrence of FRET which resulted in a fluorescent signal decrease at 470 nm and enhancement at 543 nm. The ratiometric fluorescent probe exhibited excellent selectivity to Cys over Hcy and GSH. In addition, I_543nm/I_470nm of the probe for cysteine displayed a good linear relationship in the range of 5.0 × 10^-7-1.0 × 10^-4 mol L^-1, and the detection limit was 2.0 × 10^-7 mol L^-1. Furthermore, the probe showed low cell toxicity and was successfully applied to the confocal imaging of cysteine in HepG2 cells using dual emission channels.

Full text of DOI:10.1039/c9nj03375k

View Article Online
View Journal
NJC
New Journal of Chemistry
Accepted Manuscript
A journal for new directions in chemistry
This article can be cited before page numbers have been issued, to do this please use: Y. Bai, M. Wu, Q.
Ma, C. Wang, J. Sun, M. Tian and J. Li, New J. Chem., 2019, DOI: 10.1039/C9NJ03375K.
Volume 42
This is an Accepted Manuscript, which has been through the
Number
6
21 March 2018
Pages 3963-4776
Royal Society of Chemistry peer review process and has been
accepted for publication.
NJC
New Journal of Chemistry
rsc.li/njc
A journal for new directions in chemistry
Accepted Manuscripts are published online shortly after acceptance,
before technical editing, formatting and proof reading. Using this free
service, authors can make their results available to the community, in
citable form, before we publish the edited article. We will replace this
Accepted Manuscript with the edited and formatted Advance Article as
soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes to the
text and/or graphics, which may alter content. The journal’s standard
Terms & Conditions and the Ethical guidelines still apply. In no event
shall the Royal Society of Chemistry be held responsible for any errors
or omissions in this Accepted Manuscript or any consequences arising
from the use of any information it contains.
ISSN 1144-2546
PAPER
Chechia Hu, Tzu-Jen Lin et al.
Yellowish and blue luminescent graphene oxide quantum dots
prepared via a microwave-assisted hydrothermal route using
H2O2 and KMnO4 as oxidizing agents
rsc.li/njc

Page 1 of 29
New Journal of Chemistry
1
2
3
4
5
6
7
8
9
View Article Online
DOI: 10.1039/C9NJ03375K
1 A FRET-based ratiometric fluorescent probe for highly
2 selective detection of cysteine based on a coumarin-rhodol
3 derivative
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
5
Yu Baia, Ming-Xia Wua, Qiu-Juan Maa,* Chun-Yan Wanga, Jing-Guo Suna, Mei-Ju Tiana,
,
Jian-Sheng Lib,*
6
a School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou 450046, PR China
b Collaborative Innovation Center for Respiratory Disease Diagnosis and Treatment &
Chinese Medicine Development of Henan Province, Henan University of Chinese Medicine,
Zhengzhou 450046, PR China
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
*Corresponding author, E-mail: maqiujuan104@126.com (Q. J. Ma); li_js8@163.com (J. S. Li);
Tel: +86-371-65676656; Fax: +86-371-65680028.
1

New Journal of Chemistry
Page 2 of 29
1
2
3
4
5
View Article Online
DOI: 10.1039/C9NJ03375K
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
Abstract
6
7
8
9
Cysteine, as an important amino acid in the human body, plays a vital role in people's
normal life activities. In this paper, a ratiometric fluorescent probe for detecting cysteine was
designed and synthesized based on the fluorescence resonance energy transfer (FRET)
process. In this FRET system, a coumarin derivative was used as the energy donor and a
rhodol fluorophore was choosen as the energy receptor which was modified with an acrylate
group as a cysteine recognition unit. In the absence of cysteine, the rhodol receptor was in the
non-fluorescent lactone state and FRET process was inhibited. Upon addition of cysteine, the
closed spirolactone form was converted to a conjugated fluorescent xanthenes form to induce
the occurrence of FRET which resulted in a fluorescent signal decrease at 470 nm and
enhancement at 543 nm. The ratiometric fluorescent probe exhibited excellent selectivity to
Cys over Hcy and GSH. In addition, I543 nm/I470 nm ofthe probe for cysteine displayed a good
linear relationship in the range of 5.0×10-7-1.0×10-4 mol·L-1, and the detection limit was
2.0×10-7 mol·L-1. Furthermore, the probe showed low cell toxicity and had been successfully
applied to the confocal imaging of cysteine in HepG2 cells by dual emission channels.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Keywords: ratiometric fluorescent probe; cysteine; FRET; coumarin-rhodol; cell imaging
2

Page 3 of 29
New Journal of Chemistry
1
2
3
4
5
View Article Online
DOI: 10.1039/C9NJ03375K
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
1. Introduction
6
7
8
9
Cysteine is an important biothiol, a common amino acid in the body, which plays an
important role in reversible redox reactions, cell detoxification and metabolism.1,2 The high or
low content of the product may cause adverse reactions in the organism. For example, the loss
of cysteine leads to reduced hematopoiesis, hair pigmentation, skin development damage and
cancer.3-6 Increased cysteine levels can cause severe neurotoxicity and cardiovascular
disease.7-9 Therefore, the development of efficient and reliable methods for the detection of
cysteine is of great significance for the early diagnosis and treatment of diseases.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The methods for detecting cysteine in recent years include high-performance liquid
chromatography (HPLC),10,11 capillary electrophoresis (CE),12,13 ultraviolet-visible
spectroscopic spectroscopy (UV-Vis),14 and Fourier transform infrared (FTIR)
spectrophotometry,15 fluorescence spectroscopy.16,17 Among them, the fluorescent probe has
the advantages of high sensitivity, good selectivity, easy manipulation, no damage to the test
sample, and can be combined with fluorescence imaging technology for the in situ detection
of Cys in living cells, tissues and organisms. Real-time imaging and monitoring of related
biological processes have become an effective and reliable means of detection.18-22
Fluorescent probes currently used for cysteine often utilize nucleophilicity of thiol group or
high transition metal affinity of thiol group. The detection mechanisms involve Michael
addition reaction,23,24 Michael addition and intramolecular cyclization reaction,25-28 cyclization
with aldehyde, 29,30 cleavage of sulfonamide and sulfonate ester,31,32 cleavage of disulfide,33
cleavage of Se-N bond,34 meal complexes-displace coordination35,36 and others37-39
.
Nevertheless, most of the reported fluorescent probes are used to detect cysteine based on
3

New Journal of Chemistry
Page 4 of 29
1
2
3
4
5
View Article Online
DOI: 10.1039/C9NJ03375K
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
fluorescence enhancement or quenching at single emission wavelength, which may be
6
7
8
9
influenced by various variable factors including excitation intensity, emission collection
efficiency, probe concentration and environmental effects.40,41 In contrast, ratiometric
fluorescent probes could be applied to resolve this problem, which can offer a built-in
calibration by simultaneously measuring fluorescence intensities at two different emission
wavelengths.42,43 Fluorescence resonance energy transfer (FRET) process is widely used in
the construction of ratiometric fluorescent probes because of its large pseudo-Stokes shift and
small spectral overlap, which can better distinguish the dual emission wavelengths.44,45 But,
only few fluorescence resonance energy transfer (FRET)-based ratiometric fluorescent probe
for detecting cysteine have been reported.46,47 Therefore, FRET-based ratiometric fluorescent
probes for highly selective detection of cysteine are extremely demanded for further
researching the roles of cysteine.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In this article, a FRET-based ratiometric fluorescent probe for highly selective detection of
cysteine was designed and synthesized. A coumarin derivative with excellent optical
properties and high fluorescence quantum yield was used as an energy donor, a rhodol
derivative was applied as an energy acceptor, and an acrylate group was utilized as a cysteine
recognition group. When there is no cysteine, the rhodol energy receptor is in a
non-fluorescent spirolactone form and the FRET process of the probe remains off. So, free
probe displays inherent blue fluorescence of coumarin chromophore. However, in the
presence of cysteine the probe undergoes a Michael addition and intermolecular cyclization
reaction, and the closed spirolactone form was converted to a conjugated fluorescent xanthene
form to trigger the occurrence of FRET which resulted in a fluorescent signal decrease at 470
4

Page 5 of 29
New Journal of Chemistry
1
2
3
4
5
6
7
8
9
View Article Online
DOI: 10.1039/C9NJ03375K
89
90
91
92
93
94
95
96
97
98
99
nm and enhancement at 543 nm. The ratiometric fluorescent probe exhibited highly selectivity
toward cysteine over Hcy and GSH. Moreover, the probe showed low cell toxicity and had
been successfully applied to the confocal imaging of cysteine in HepG2 cells by dual
emission channels.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2. Experimental
2.1. Materials and instruments
4-(Diethylamino)salicylaldehyde, diethyl malonate, m-diphenol, phthalic anhydride,
trifluoroacetic acid, 4-diaminopyridine (DMAP), acryloyl chloride were purchased
from Heowns Biochemical Technology Company.
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and
m-hydroxyphenylpiperazine were purchased from Energy Chemical (Shanghai, China).
Cysteine (Cys) and homocysteine (Hys) were purchased from TCI (Shanghai) Development
Company. Glutathione (GSH) is purchased from Aladdin Reagent Company. Threonine (Thr),
leucine (Leu), methionine (Met), valine (Val), phenylalanine (Phe), serine (Ser), asparagine
(Asn), tryptophan (Trp), tyrosine (Tyr), glutamine(Gln), lysine (Lys), isoleucine (Ile), alanine
(Ala), histidine (His), aspartic acid (Asp), arginine (Arg), proline (Pro), glutamic acid (Glu)
and glycine (Gly) were obtained from Shanghai Lanji Science and Technology Development
Company. Anhydrous aluminum chloride and n-hexane were purchased from Tianjin Sailboat
Chemical Reagent Technology Company. Nitrobenzene was purchased from Tianjin Damao
Chemical Reagent Factory. Hexahydropyridine was purchased from Shanghai Pharmaceutical
Reagent Company of China Pharmaceutical Group. Triethylamine was purchased from
Tianjin Fuchen Chemical Reagent Factory. The solvents used in the high performance liquid
100
101
102
103
104
105
106
107
108
109
110
5

New Journal of Chemistry
Page 6 of 29
1
2
3
View Article Online
chromatography (HPLC) experiments were all chromatographically pure and purchasDeOdI:f1r0o.1m039/C9NJ03375K
Tianjin Siyou Fine Chemicals Company. All other chemical reagents were analytically pure
reagents, purchased from commercial suppliers and used directly in the experiment without
further purification. Thin layer chromatography was performed using silica gel 60 F254, and
column chromatography was conducted on silica gel (200-300 mesh), both of which were
purchased from China Qingdao Ocean Chemical. Water is purified by SZ-93 automatic
double pure water distiller (Shanghai Yarong Biochemical Instrument Factory).
4
5
6
7
8
9
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The NMR spectrum was recorded with Bruker's DRX-500 spectrometer using
tetramethylsilane (TMS) as an internal standard. Mass spectra were obtained on an Agilent
Technologies 6420 Triple Quad LC/MS high resolution mass spectrometer. All fluorescence
tests were performed on a Hitachi F-7000 fluorescence spectrophotometer with a 1 cm quartz
absorption cell with an excitation wavelength of 418 nm and an entrance and exit slit of 10
nm. The UV-visible absorption spectrum was measured by an EVOLUTION 260 BIO
UV-Vis spectrophotometer with a 1 cm quartz absorption cell. The pH was measured by a pH
meter (METTLER TOLEDO Fiveeasy Plus). The DF-101S collector-type
constant-temperature heating magnetic stirrer produced by Gongyi City Yuhua Instrument
Company and the MS-PB magnetic stirrer manufactured by Shanghai Yuhuai Instrument
Company were used in the synthesis process. High performance liquid chromatograms were
obtained with an UltiMate 3000 high performance liquid chromatograph equipped with an
XBP-C18 column (5 µm, 4.6×250 mm). Fluorescence imagings of living cells were recorded
by an Olympus FV-1200 single photon laser confocal microscope. Data processing is mainly
obtained in SigmaPlot software. The data obtained by fluorescence spectrophotometry and
6

Page 7 of 29
New Journal of Chemistry
1
2
3
4
5
6
7
8
9
View Article Online
DOI: 10.1039/C9NJ03375K
133
134
135
136
137
138
139
140
141
142
143
144
UV-visible spectrophotometry were measured in 0.01 mol/L PBS buffer (CH3CN/water = 6:4,
V/V, pH =7.40). In addition to the fluorescence data obtained by time scanning, all other
fluorescence and absorption data were recorded at 60 min after addition of cysteine at room
temperature.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2.2. Syntheses
The synthetic route for FRET-based ratiometric fluorescence probe 1 for highly selective
detection of cysteine is shown in Scheme 1. The probe 1 uses a coumarin-rhodol fluorescence
resonance energy transfer system as a mechanism. Fluorescence emission spectrum of
coumarin energy donor (compound 5) efficiently overlaps with the UV-vis absorption
spectrum of rhodol energy acceptor (compound 3), indicating that FRET process would occur
to induce the appearance of acceptor emission concomitant with disappearance of donor
emission (Figure 1).
145
146
Scheme 1 Synthesis of FRET-based ratiometric fluorescent probe 1: (a) nitrobenzene,
anhydrous AlCl3, 84%; (b) m-hydroxyphenylpiperazine, CF3COOH, 75%; (c) diethyl
147
7

New Journal of Chemistry
Page 8 of 29
1
2
3
4
5
6
7
View Article Online
148
149
malonate, hexahydropyridine, 81%; (d) Ⅰ. NaOH, Ⅱ. HCl , 75%; (e) EDC, DMAP,D4O3I:%10;.1(0f3)9/C9NJ03375K
anhydrous CH2Cl2, triethylamine, acryloyl chloride, 61%.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
150
151
Figure 1 Normalized emission spectra of donor derivative 5 (5.0 µM ) and normalized
absorption spectra of acceptor 3 (5.0 µM ) in 0.01 M PBS buffer (CH3CN/water = 6:4, V/V,
152
153
154
155
156
157
158
159
160
pH=7.40). The medium dash (̶ ̶ ̶ ) and solid line (—) represent normalized absorption of
compound 3 and normalized emission spectra of compound 5, respectively.
Synthesis of compound 2. The compound 2 is synthesized according to the reported
document.45 Under a nitrogen stream, resorcinol (0.55 g, 5 mmol) and phthalic anhydride
(0.74 g, 5 mmol) were dissolved in 30 ml of nitrobenzene, and then anhydrous AlCl3 (1.47 g,
11mmol) was added, and the reaction was stirred at room temperature for 12 h. The reaction
mixture was poured into a two-phase solution of vigorously stirred 30 ml n-hexane and 40 ml
0.5 M HCl. After the reaction was stirred for 2 h, an orange-yellow precipitate was obtained
8

Page 9 of 29
New Journal of Chemistry
1
2
3
4
5
6
7
8
9
View Article Online
161
and filtered to give a crude product. Then The product was purified throughDOcIo: 1l0u.m103n9/C9NJ03375K
chromatograph using dichloromethane/methanol (15:1, v/ v) as the eluent to obtain compound
2 as an orange solid (1.08 g, 84%).1H NMR (500 MHz, CD3OD), δ(ppm): 8.06 (1H, d, J = 7.7
Hz), 7.65 (1H, t, J = 7.5 Hz), 7.59 (1H, t, J = 7.5 Hz), 7.34 (1H, d, J = 7.5 Hz), 6.93 (1H, d, J
= 8.8 Hz), 6.31 (1H, d, J = 2.0 Hz), 6.21(1H, dd, J = 8.8 Hz, 2.0 Hz).
162
163
164
165
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
166
167
168
169
170
171
172
173
174
Synthesis of Compound 3. The compound 3 is synthesized according to the reported
document.45 Compound 2 (1.03 g, 4 mmol) was dissolved in 20 ml of CF3COOH and then
m-hydroxyphenylpiperazine (0.71 g, 4mmol) was also added into the above mixture. This
reaction mixture was heated to reflux for 36 h, then the solvent was removed under reduced
pressure. It was further purified by column chromatograph using dichloromethane/methanol
(5:1, v/v) to obtain a red solid compound 3 (1.21 g, 75%). 1H NMR (500 MHz, DMSO-d6),
δ(ppm): 10.22 (1H, s), 7.99 (1H, d, J = 7.7 Hz), 7.80-7.77 (1H, m), 7.72-7.69 (1H, m), 7.24
(1H, d, J = 7.6 Hz), 6.89 (1H, d, J = 2.3 Hz), 6.76 (1H, dd, J = 8.9 Hz, 2.3 Hz), 6.68 (1H, s),
6.58-6.55 (3H, m), 3.44 (4H, d, J = 5.3 Hz), 3.21 (4H, d, J = 4.8 Hz).
175
176
177
178
179
180
181
Synthesis of compound 4. Synthesis of compound 4 according to reported literature. 45
4-(Diethylamino)salicylaldehyde (0.97 g, 5 mmol) was dissolved in 25 ml CH3CH2OH, then
diethyl malonate (0.96 g, 6 mmol) and 0.5 ml hexahydropyridine were added. The reaction
mixture was stirred under reflux for 2 h, diluted with 50 ml water, extracted with ethyl acetate
(50 ml × 3), and dried over anhydrous MgSO4. The solvent was removed under reduced
pressure and the crude product was purified by column chromatography using petroleum
ether/ethyl acetate ( 3:1, v/v) to get an orange compound 4 (1.16 g，81%). 1H NMR (500MHz,
9

New Journal of Chemistry
Page 10 of 29
1
2
3
4
5
6
7
8
9
View Article Online
DOI: 10.1039/C9NJ03375K
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
CDCl3), δ(ppm): 8.40 (1H, s), 7.33 (1H, d, J =8.9 Hz), 6.57 (1H, dd, J = 8.9 Hz, 2.2 Hz), 6.43
(1H, d, J = 2.1 Hz), 4.34 (2H, q, J = 7.1 Hz), 3.42 (4H, q, J = 7.1 Hz), 1.36 (3H, t, J = 7.1 Hz),
1.20 (6H, t, J = 7.1 Hz).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of compound 5. Synthesis of compound 5 according to reported literature.
45Compound 4 (1.05 g, 4 mmol) was dissolved in 40 ml ethanol and then 40 ml 0.5 M NaOH
was added. This mixture was stirred at room temperature for 12 h. The solvent was
evaporated under reduced pressure to give a solid. The solid was dissolved in 5 ml water, and
the pH of the solution was acidified with 1M HCl to give a precipitate. When the precipitate
was filtered, a red-brown solid compound 5 (0.78 g, 75%) was obtained. 1H NMR (500MHz,
CDCl3), δ(ppm): 12.30 (1H, s), 8.64 (1H, s), 7.44 (1H, d, J = 9.1 Hz), 6.69 (1H, dd, J = 9.1 Hz,
2.4 Hz), 6.51 (1H, d, J = 2.4 Hz), 3.47 (4H, q, J = 7.2 Hz), 1.24 (6H, t, J = 7.2 Hz).
Synthesis of compound 6. Under nitrogen protection, compound 3 (0.40 g, 1 mmol) was
dissolved in 60 ml anhydrous dichloromethane/anhydrous N,N-dimethylformamide (5:1, v/ v).
Then DMAP (0.06 g, 0.5 mmol) and EDC (0.19 g, 1 mmol) were added. Then the mixture
was stirred at room temperature for 30 min. Compound 5 (0.31 g, 1.2 mmol) was added and
stirred overnight. The reaction solution was evaporated under reduced pressure to give a crude
product. Then it was further purified by column chromatograph using
dichloromethane/methanol (20:1, v/ v) as the eluent to get a red compound 6 (0.28 g, 43%).
1H NMR (500 MHz, DMSO-d6), δ(ppm): 10.10 (1H, s), 8.00-7.97 (2H, m), 7.77 (1H, d, J =
7.5 Hz), 7.71 (1H, d, J = 7. 5 Hz), 7.49 (1H, d, J = 8.9 Hz), 7.25 (1H, d, J = 7.6 Hz), 6.81 (1H,
s), 6.74 (2H, d, J = 9.0 Hz), 6.66 (1H, s), 6.54 (4H, d, J = 7.0 Hz), 3.69 (2H, s), 3.45-3.42 (6H,
m), 3.32 (2H, d, J = 1.1 Hz), 3.25 (2H, s), 1.12 (6H, t, J = 7.0 Hz). MS (ESI) m/z: 644.1000
10

Page 11 of 29
New Journal of Chemistry
1
2
3
4
View Article Online
DOI: 10.1039/C9NJ03375K
204
(M+H)+, 666.1000 (M+Na)+.
5
6
7
8
9
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
Synthesis of compound 1. Compound 6 (0.64 g, 1mmol) was dissolved in 20 ml anhydrous
dichloromethane followed by addition of 1.4 ml triethylamine, then acryloyl chloride (0.18 g,
2 mmol) was added slowly. After half an hour of reaction at 0℃, the reaction was stirred at
room temperature for 12 h. Subsequently, the solvent was evaporated under reduced pressure.
The crude product was further purified by column chromatograph using
dichloromethane/methanol (30:1, v/ v) as the eluent to obtain a yellow compound 1 (0.43 g,
61%). 1H NMR (500 MHz, CDCl3), δ(ppm): 7.98 (1H, d, J = 7.6 Hz), 7.86 (1H, s), 7.64 (1H, t,
J = 7.4 Hz), 7.58 (1H, t, J = 7.4 Hz), 7.27 (1H, d, J = 8.8 Hz), 7.15 (1H, d, J = 7.6 Hz),
7.08-7.07 (1H, m), 6.80-6.76 (2H, m), 6.68 (1H, d, J = 1.9 Hz), 6.64-6.56 (4H, m), 6.44 (1H,
d, J = 1.9 Hz), 6.28 (1H, dd, J = 17.3 Hz, 10.5 Hz), 6.01 (1H, d, J = 10.5 Hz), 3.86 (2H, s),
3.54 (2H, s), 3.40 (4H, q, J = 7.0 Hz), 3.30 (4H, d, J = 16.6 Hz), 1.19 (6H, t, J = 7.0 Hz). 13C
NMR (125 MHz, CDCl3), δ(ppm): 169.29, 165.02, 163.88, 159.09, 157.26, 152.90, 152.52,
152.12, 151.94, 151.71, 151.69, 145.39, 135.00, 133.17, 129.86, 129.72, 128.95, 128.70,
127.45, 126.50, 124.94, 123.96, 117.11, 116.82, 115.76, 112.33, 110.17, 109.35, 109.21,
107.66, 102.35, 96.83, 82.60, 48.40, 47.95, 46.77, 44.87, 41.88, 12.33. MS (ESI) m/z:
698.2000 (M+H)+, 702.2000 (M+Na)+. In the DEPT135 spectrum, the chemical shift of 82.60
corresponds to the carbonyl carbon of spirolactone, indicating that compound 1 was in a
spirolactone form.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2.3. Cytotoxicity assay
Cytotoxicity is an important indicator of the performance of fluorescent probe bioimaging
applications. In order to detect the cytotoxicity of the probe,
11

New Journal of Chemistry
Page 12 of 29
1
2
3
4
View Article Online
DOI: 10.1039/C9NJ03375K
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT) colorimetric method was
used. The selected cells were HepG2 cells (liver cancer cells). First, the HepG2 cells were
cultured in 10% fetal bovine serum medium, about 1×104 cells were seeded in each well of a
96-well plate and the total volume per well was controlled at 100 µL . After the HepG2 cells
was placed in an incubator containing 5% CO2 at 37℃ for 24 h, the medium was aspirated.
Then, the HepG2 cells were incubated with different concentrations of compound 1 and
compound 6 with fresh medium for 4 h. After that, the medium was removed from this
96-well plate and added to the fresh medium for 24 h. The HepG2 Cells were incubated with
the fresh medium (100µL ) containing MTT (10µL , 5 mg·ml-1) for 4 h. At last, the supernatant
in the 96-well plate was aspirated and 150 μL DMSO was participated, shaked for 10 min.
Then the absorbance at 490 nm was measured with a microplate reader and cell viability was
estimated by A/A0× 100% (A and A0 are the absorbance of experimental group and control
group, respectively).
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2.4. Confocal imaging in living cells
The HepG2 cells were cultured on the laser confocal culture dishes at 37℃ for 24 h to
ensure good cell growth, and then washed three times with Dulbecco s phosphate buffered
̕
saline (DPBS). In a control experiment, HepG2 cells were pretreated with 1 mM
N-methylmaleimide (sulfhydryl masking agent) for 40 min, washed three times with DPBS.
Then HepG2 cells were incubated with 10 μM probe 1 for 45 min, rinsed with DPBS three
times and imaged. In the experimental group of imaging endogenous Cys, HepG2 cells were
treated with 10 μM probe 1 for 45 min, after which the cells were washed with DPBS three
times and imaged. In the experimental group of imaging exogenous Cys, HepG2 cells were
12

Page 13 of 29
New Journal of Chemistry
1
2
3
4
5
6
7
8
9
View Article Online
DOI: 10.1039/C9NJ03375K
248
249
250
251
252
253
incubated with 10 µM probe 1 for 45 min and washed three times with DPBS, after which the
cells were incubated with 0.1 mM Cys for another 45 min, washed three times with DPBS and
imaged. Confocal fluorescence images were observed by an Olympus FV1200-MPE
multiphoton confocal microscope with 60× objective lens.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3. Results and discussion
3.1. Spectroscopic analytical performance of probe 1 towards Cys
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
In presence of 100 μM cysteine, the probe can exhibit better fluorescence sensing for
cysteine (Figure S1). So, 60% organic solvents in buffer solution was used in the experiment.
To study the fluorescence sensing properties of probe 1 for Cys, we investigated the change of
fluorescence response of probe 1 (10 μM) to 0.01 M PBS buffer (CH3CN: water= 6:4, v/v, pH
= 7.40) containing different amounts of Cys (Figure 2). From Figure 2, when there is no
cysteine, probe 1 exhibited strong fluorescence with an emission peak at 470 nm. However, in
the presence of incremental Cys (0-100 μM), the initial fluorescence at 470 nm decreased
gradually, a new fluorescence emission peak appeared at 543 nm and increased progressively,
permitting a ratiometric fluorescence response for Cys. It is remarkable that the large
emission shift (Δλ=73 nm) results in two well-resolved emission bands for the probe, which
would be beneficial for dual-channel imaging of Cys in biological samples with less
cross-talk observed. The notable change in fluorescence spectra should be attributed to
Cys-induced occurrence of FRET between the donor coumarin and acceptor rhodol. FRET
efficiency is an important parameter of the FRET dye, which indicates the energy transfer
efficiency between the donor and the acceptor. The fluorescence emission intensities of
compound 5 (10 μM) and the reaction product of fluorescent probe 1 (10.0 µM) with cysteine
13

New Journal of Chemistry
Page 14 of 29
1
2
3
4
5
6
7
8
9
View Article Online
DOI: 10.1039/C9NJ03375K
270
271
272
(100 µM) at 470 nm were 8222 and 862 (Figure S2), so the fluorescence resonance energy
transfer efficiency was estimated to be 89.5% according to Energy Transfer Efficiency (ETE)
= [(fluorescence of donor-fluorescence of donor in cassette)/fluorescence of donor]×100%.48
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
273
274
Figure 2 One-photon fluorescence spectra of probe 1 (10.0 µM) in the presence of various
concentrations of cysteine: 0, 0.5, 0.7, 1.0, 3.0, 5.0, 7.0, 10, 20, 30, 50, 70, 100 μM from 1 to
13 (λex =418 nm).
275
276
277
278
279
280
281
282
283
284
We further studied the UV-vis absorption spectra of probe 1 (10 μM), the reaction mixture
of fluorescent probe 1 (10 μM) with cysteine (100 μM), and compound 6 (10 μM) (Figure 3).
From Figure 3, Probe 1 has a maximum absorption at 407 nm, which corresponds to
absorption of the coumarin donor, 49 and compound 6 absorbs at both 407 nm and 511 nm. In
the presence of cysteine, no obvious absorption changes of the donor occurred, while a new
aborption peak at 511 nm belonging to the conjugated xanthene form of the acceptor
emerged.50 The above results indicates that the reaction of the probe 1 with cysteine yields the
compound 6.
14

Page 15 of 29
New Journal of Chemistry
1
2
3
4
View Article Online
DOI: 10.1039/C9NJ03375K
5
6
7
8
9
10
11
12
13
14
15
16
17
285
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
286
287
288
289
290
Figure 3 Absorption spectra of fluorescent probe 1 (10 µM), compound 6 (10 µM), and the
reaction mixture of fluorescent probe 1 (10 µM) with cysteine (100 µM). The dotted line (···),
solid line (—), and medium dash (̶ ̶ ̶ ) represent fluorescent probe 1, compound 6 and the
reaction product of fluorescent probe 1 with cysteine, respectively. Inset: color changes in
probe 1 upon addition of cysteine.
291
292
293
294
295
296
297
298
299
300
3.2. Principle of operation and the basis of quantitative assay
In order to investigate the linear response of the ratio value of the emission intensities at
543 nm and 470 nm (I543nm/I470nm) toward Cys, we chose different concentrations of Cys for
experiments. When the concentration of cysteine was 5.0×10-7-1.0×10-4 mol·L-1 (Figure 4),
the ratio of I543nm/I470nm was linear with the concentration of Cys. The linear regression
equation was I543nm/I470nm = 0.0763 + 0.0507×106×C (R = 0.9997), C is the concentration of
cysteine, and R is the linear correlation coefficient. The detection limit was calculated by
3SB/m, SB is the standard deviation of the fluorescence intensity measured 10 times for the
blank solution, and m is the slope of the calibration curve.49 The limit detection of probe 1 for
cysteine was 2.0× 10-7 mol·L-1, which is much lower than previously reported ratiometric
15

New Journal of Chemistry
Page 16 of 29
1
2
3
4
5
6
7
View Article Online
DOI: 10.1039/C9NJ03375K
301
302
fluorescent probe for Cys.46, 51-53 The results indicate that probe 1 can be used for highly
sensitive quantitative detection of cysteine.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
303
304
Figure 4. Fluorescence intensity ratio (I543 nm /I470 nm) of probe 1 (10 µM) as a function of the
concentration of cysteine from 0.5 µM to 100 µM. The inset exhibits the corresponding
fluorescence intensity ratio (I543 nm /I470 nm) of probe 1 (10 µM) as a function of the concentration
of cysteine from 0.5 µM to 5.0 µM.
305
306
307
308
309
310
311
312
313
314
315
316
317
The response mechanism of probe 1 to cysteine may be ascribed to the reaction of probe 1
with Cys to produce compound 6 (Scheme 2). As displayed in Scheme 2, in the absence of
cysteine, the rhodol receptor was in the non-fluorescent lactone state and FRET process was
inhibited. Upon addition of cysteine, a Michael addition reaction was taken place between
acryloyl group of probe 1 and thiol group of Cys to generate thioether compound 7.
Subsequently, compound 7 underwent an intramolecular cyclization to produce compound 6
accompanied by the release of the cyclization product 8. Thus, in the presence of cysteine, the
closed spirolactone form was converted to a conjugated fluorescent xanthenes form to induce
the occurrence of FRET. In order to further verify the reaction mechanism of probe 1 for
cysteine, we performed HPLC on probe 1, reaction mixture of probe 1 with Cys and
16

Page 17 of 29
New Journal of Chemistry
1
2
3
4
5
6
7
8
9
View Article Online
DOI: 10.1039/C9NJ03375K
318
319
320
321
322
323
324
325
326
327
compound 6 (Figure 5). As shown in Figure 5, probe 1 peaked at 6.65 min, the reaction
mixture of probe 1 with cysteine showed a peak after 2.98 min of injection, which was
consistent with the retention time of compound 6 in the HPLC. The ESI-MS data of reaction
mixture of probe 1 with Cys for 10 min displayed signal at 642.2215 and 819.0283,
respectively (Figure S3). As illustrated in Figure S3, the signal at 642.2215 was consistent
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
with the structure [compound 6 ̶
H]+ and the signal at 819.0283 was attributed to the structure
[compound 7 + H]+. Simultaneously, in the ultraviolet absorption spectrum property, the
maximum absorption wavelength of the compound 6 and the reaction mixture of probe 1 with
cysteine was identical. Therefore, it can be proved that the response mechanism of probe 1 to
cysteine was presumed to be correct.
328
329
Scheme 2. Proposed possible mechanism of the response of compound 1 to cysteine
17

New Journal of Chemistry
Page 18 of 29
1
2
3
4
View Article Online
DOI: 10.1039/C9NJ03375K
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
330
331
Figure 5. HPLC profifiles of (A) compound 1 (10 μM), (B) compound 6 (10 μM) and (C) the
332
reaction mixture of compound 1 (10 μM) with cysteine (100 μM). HPLC conditions:
333
334
335
1.0 mL/min total flow rate, Agela Technologies Venusil XBP-C18: 5 μm, 4.6 × 250 mm
column, isocratic elution with acetonitrile at flow rate 0.8 mL/min and water at flow rate 0.2
mL/min, detected at 407 nm.
336
3.3. Time-dependent responses and effect of pH
18

Page 19 of 29
New Journal of Chemistry
1
2
3
4
5
6
7
8
9
View Article Online
DOI: 10.1039/C9NJ03375K
337
338
339
340
341
342
We further investigated the time response of probe 1 (10 μM) under conditions of addition of
Cys (100 μM) (Figure S4). Figure S4 showed the trend of the value of I543 nm/I470 nm over time.
When Cys (100 μM) was added, the value continues to increase and reached a maximum and
saturate at 60 min. The experimental results showed that probe 1 is stable under the
experimental conditions, and the detection of cysteine can be completed within 60 min. In this
study, an assay time of 60 min was chosen as the optical measurement condition.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
343
344
345
346
347
In order to test the sensitivity of synthesized probe 1, we studied changes in the I543 nm/I470
nm values of synthesized probe 1 (10 μM) before and after adding Cys (100 μM) at different
pH values (Figure 6). As depicted in Figure 6, the probe 1 has good sensitivity to the Cys at a
pH range of 6.00-11.50.Therefore, experimental results showed that probe 1 can work in a
large pH range and can be used for biological detection.
348
349
350
351
Figure 6. Effect of pH on the ratio of fluorescence intensity (I543 nm/I470 nm) of 10 µM probe 1
in the absence (filled circles) and presence of 100 µM cysteine (clear circles). All data were
obtained at various pH values (pH 2.00-11.50).
19

New Journal of Chemistry
Page 20 of 29
1
2
3
4
5
6
7
8
9
View Article Online
DOI: 10.1039/C9NJ03375K
352
353
354
355
356
357
358
359
360
361
3.4. Selectivity
An important indicator for evaluating the performance of fluorescent probe for target sensing
is selectivity. We studied the fluorescence response of probe 1 to cysteine and other related
substances at pH 7.40 (Figure 7A). As shown in Figure 7A, in the presence of cysteine probe
1 showed significant fluorescence enhancement, while the addition of other substances had
little effect on the fluorescence of probe 1. To investigate the potential applications of probes
in complex biological samples, we also investigated the effect of coexistence of other
substances and cysteine on the sensitivity of fluorescent probe to cysteine at a pH of 7.40
(Figure 7B). From Figure 7B, the probe still maintains a fluorescent response to cysteine in
the presence of other related substances.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
362
20

Page 21 of 29
New Journal of Chemistry
1
2
3
4
5
6
7
8
9
View Article Online
DOI: 10.1039/C9NJ03375K
363
364
365
366
367
368
369
370
371
372
373
Figure 7. (A) Fluorescence response of probe 1 (10 μM) toward Cys and other substances at
pH 7.40: (1)blank, (2) Thr, (3) Leu, (4) Met, (5) Val, (6) Phe, (7) Ser, (8) Asn, (9) Trp, (10)
Tyr, (11) Gln, (12) Lys, (13) Ile, (14) Ala, (15) His, (16) Asp, (17) Arg, (18) Pro, (19) Glu,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(20) Gly, (21) F-, (22) CO3 , (23) CH3COO-, (24) S2O32-, (25) NO2 , (26) SO4 , (27) Cl-, (28)
2-
-
2-
-
2-
NO3 , (29) I-, (30) Br-, (31) SCN-, (32) SO3 , (33) Hcy, (34) GSH, (35) Cys; (B) Fluorescence
response of probe 1 (10 μM) toward Cys in the presence of other substances at pH 7.40: (1)
Thr, (2) Leu, (3) Met, (4) Val, (5) Phe, (6) Ser, (7) Asn, (8) Trp, (9) Tyr, (10) Gln, (11) Lys,
(12) Ile, (13) Ala, (14) His, (15) Asp, (16) Arg, (17) Pro, (18) Glu, (19) Gly, (20) F-, (21)
2-
2-
-
2-
-
CO3 , (22) CH3COO-, (23) S2O3 , (24) NO2 , (25) SO4 , (26) Cl-, (27) NO3 , (28) I-, (29) Br-,
2-
(30) SCN-, (31) SO3 , (32) Hcy, (33) GSH, (34) Cys. The concentration of all substances
added to probe 1 is 100 μM.
374
375
376
377
378
3.5. Cytotoxicity assays and confocal imaging in living cells
We tested cytotoxicity of probe 1 and compound 6 at different concentrations (0, 2, 4, 8, 16
μM) against HepG2 cells by MTT assay (Figure 8). As depicted in Figure 8, when probe 1
and compound 6 were present, cell viability reached 80% or more, which indicates that probe
1 and compound 6 were almost non-toxic to HepG2 cells.
21

New Journal of Chemistry
Page 22 of 29
1
2
3
4
View Article Online
DOI: 10.1039/C9NJ03375K
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
379
380
Figure 8. MTT assay of HepG2 cells in the presence of different concentrations of compound
1 (a) and compound 6 (b) (0, 2, 4, 8, 16 µM) for 24 h at 37 ℃, respectively.
381
382
383
In order to demonstrate the practicality of probe 1 in biology, we performed laser confocal
fluorescence imaging of cysteine in HepG2 cells with fluorescent probe 1 (Figure 9). HepG2
22

Page 23 of 29
New Journal of Chemistry
1
2
3
4
5
6
7
8
9
View Article Online
384
cells were seeded in three 35mm laser confocal culture dishes and incubated for 24 h.DTOhI:e10.1039/C9NJ03375K
HepG2 cells was pretreated with 1 mM N-methylmaleimide (sulfhydryl masking agent) for 40
min, and incubated with 10 μM probe 1 medium for 45 min for fluorescence imaging, it was
found that the blue channel has obvious fluorescence (Figure 9b), and the red channel has
almost no fluorescence (Figure 9 c). In the control experiment, HepG2 cells were incubated in
a medium containing 10 μM probe 1 for 45 min for fluorescence imaging. The results showed
that the blue channel showed weak fluorescence (Figure 9 f) and the red channel was more
obvious fluorescence (Figure 9 g). Simultaneously, HepG2 cells were incubated with 10 μM
probe 1 for 45 min, then incubated with 0.1 mM Cys for 45 min and imaged. The
experimental results exhibited that the fluorescence of the blue channel was weak (Figure 9 j),
and the fluorescence of the red channel was stronger than that without added Cys (Figure 9 k).
These phenomena indicate that the probe 1 can be used for the ratiometric dual-channel
detection of cysteine in living cells.
385
386
387
388
389
390
391
392
393
394
395
396
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
397
398
Figure 9. Laser confocal fluorescence imaging of cysteine in HepG2 cells with fluorescent
probe 1. (a) Bright field image after incubation of HepG2 cells with 1 mM
399
23

New Journal of Chemistry
Page 24 of 29
1
2
3
View Article Online
N-methylmaleimide for 40 min, and incubation of HepG2 cells with 10 μM probeDfOoIr: 110.140359/C9NJ03375K
min; (b) fluorescence image from blue channel of image (a); (c) fluorescence image from red
channel of image (a); (d) overlay image of (a), (b) and (c); (e) Bright field map after
incubation of HepG2 cells for 45 min with 10 μM probe 1; (f) fluorescence image from blue
channel of image (e); (g) fluorescence image from red channel of image (e); (h) overlay
image of (e), (f) and (g); (i) bright field images of HepG2 cells incubated with probe 1 for 45
min, then with 10 μM Cys for 45 min; (j) fluorescence image from blue channel of image (i);
(k) fluorescence image from red channel of image (i); (l) overlay image of (i), (j) and (k).
4. Conclusions
4
5
6
7
8
9
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In summary, we designed and synthesized a FRET-based ratiometric probe for detection of
Cys based on a coumarin-rhodol derivative. An acrylate group was applied as an identification
group for Cys. In the absence of cysteine, the rhodol receptor was in the non-fluorescent
lactone state and FRET process was inhibited. Upon addition of cysteine, the closed
spirolactone form was converted to a conjugated fluorescent xanthenes form to induce the
occurrence of FRET which resulted in a fluorescent signal decrease at 470 nm and
enhancement at 543 nm. The probe shows high sensitivity and selectivity for cysteine over
glutathione, homocysteine, and other related substances. In addition, the probe also has good
cell permeability and has been successfully applied for the ratiometric dual-channel detection
of cysteine in living cells.
Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgments
24

Page 25 of 29
New Journal of Chemistry
1
2
3
4
5
6
7
8
9
View Article Online
422
This work was supported by the Key Science and Technology Programme oDfOHI: e10n.1a0n39/C9NJ03375K
Province (182102310101), National Natural Science Foundation of China (Grant nos.
21807027 and 81130062), the Fundamental Research Funds for the Provincial
Universities (2014KYYWF-QN04), and Graduate Innovation Fund in Henan College
of Chinese Medicine (YJS2018A05).
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
References
1 X. Chen, Y. Zhou, X. Peng and J. Yoon, Chem. Soc. Rev., 2010, 39, 2120.
2 Y. Yang, Q. Zhao, W. Feng and F. Li, Chem. Rev., 2013, 113, 192.
3 D. M. Townsend, K. D. Tew and H. Tapiero, Biomed. Pharmacother., 2003, 57, 145.
4 K. G. Reddie and K. S. Carroll, Curr. Opin. Chem. Biol., 2008, 12, 746.
5 E. Weerapana, C. Wang, G. M. Simon, M. B. D. Dillon, D. A. Bachovchin, K. Mowen, D.
Baker and B. F. Cravatt, Nature, 2010, 468, 790.
6 A. K. Elshorbagy, V. Kozich, A. D. Smith and H. Refsum, Curr. Opin. Clin. Nutr. Metab.
Care, 2012, 15, 49.
7 M. T. Heafield, S. Fearn, G. B. Steventon, R. H. Waring, A. C. Williams and S. G. Sturman,
Neurosci. Lett., 1990, 110, 216.
8 R. Janáky, V. Varga, A. Hermann, P. Saransaari and S. S. Oja, Neurochem. Res., 2000, 25,
1397.
9 X. F. Wang and M. S. Cynader, J. Neurosci., 2001, 21, 3322.
10 W. Zhang, P. Li, Q. Geng, Y. Duan, M. Guo and Y. Cao, J. Agric. Food Chem., 2014, 62,
5845.
11 Y. Wang, C. H. Zhang, Y. H. Zheng, Y. Ge, X. Y. Yu, Anal. Lett., 2019, 52, 1487.
25

New Journal of Chemistry
Page 26 of 29
1
2
3
4
5
View Article Online
DOI: 10.1039/C9NJ03375K
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
12 A. V. Ivanov, E. D. Virus, B. P. Luzyanin and A.A. Kubatiev, J. Chromatogr. B., 2015,
6
7
8
9
1004, 30.
13 R. C. de Carvalho, T. R. D. Mathias, A. D. P. Netto, F. F. D. Marques, Electrophoresis,
2018, 39, 1613.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
14 N. Shao, J. Y. Jin, S. M. Cheung, R. H. Yang, W. H. Chan and T. Mo, Angew. Chem., 2006,
118, 5066.
15 Y. Sato, T. Iwata, S. Tokutomi and H. Kandori, J. Am. Chem. Soc., 2005, 127, 1088.
16 Y. Zhou and J. Yoon, Chem. Soc. Rev., 2012, 41, 52.
17 X. Chen, T. Pradhan, F. Wang, J. S. Kim and J. Yoon, Chem. Rev., 2012, 112, 1910.
18 X.Yang, Y. Guo and R. M. Strongin, Angew. Chem. Int. Ed., 2011, 50, 10690.
19 L. He, B. Dong, Y. Liu and W. Lin, Chem. Soc. Rev., 2016, 45, 6449.
20 L. Y. Niu, Y. Z. Chen, H. R. Zheng, L. Z. Wu, C. H. Tung and Q. Z. Yang, Chem. Soc. Rev.,
2015, 44, 6143.
21 Z. L. Lu, Y. N. Lu, C. H. Fan, X. Sun, W. Q. Shao, N. Jiang, X. Y. Gong, Y. Z. Lu, G. X.
Sun and X. C. Jiang, Sens. Actuators B, 2019, 290, 581.
22 D. J. Zhu, X. W. Yan, A. S. Ren, W. Xie and Z. H. Duan, Anal. Chim. Acta, 2019, 1058,
136.
461
462
23 P. Srivastava, R. C. Gupta and A. Misra, ChemistrySelect, 2018, 3, 12900.
24 J. Li, Y. k. Yue, F. J. Huo and C. X. Yin, Dyes Pigments, 2019, 164, 339.
463
464
25 W. Zhang, X.Y. Zhao, W. J. Gu, T. Cheng, B. X. Wang, Y. L. Jiang and J. Shen, New J.
Chem., 2018, 42, 18109.
26

Page 27 of 29
New Journal of Chemistry
1
2
3
4
View Article Online
465
26 S. Jiao, X. He, L.B. Xu, P.Y. Ma, C. M. Liu, Y. B. Huang, Y. Sun, X. H. Wang anDdODI: 1.0Q.10.39/C9NJ03375K
5
6
7
466
Song, Sens. Actuators B, 2019, 290, 47.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
467
468
27 Z. L. Lu , Y. N. Lu , C. H. Fan , X. Sun , W.Q. Shao, N. Jiang , X. Y. Gong , Y. Z. Lu , G.
X. Sun and X. C. Jiang, Sens. Actuators B, 2109, 290, 581.
469
470
28 D. G. Chen, Z. Long, Y. C. Dang and L. Chen, Dyes Pigments, 2019, 166, 266.
29 K. S Lee, T. K Kim, J. H. Lee, H. J. Kim and J. I. Hong, Chem. Commun., 2008, 46, 6173.
471
472
30 Z. G. Yang, N. Zhao, Y. M. Sun, F. Miao, Y. Liu, X. Liu, Y. H. Zhang, W. T. Ai, G. F.
Song, X. Y. Shen, X. Q. Yu, J. Z. Sun and W. Y. Wong, Chem. Commun., 2012, 48, 3442.
473
474
31 M. J. Wei, P. Yin, Y. M. Shen, L. L. Zhang, J. H. Deng, S. Y. Xue, H.T. Li, B. Guo, Y. Y.
Zhang and S. Z. Yao, Chem. Commun., 2013, 49, 4640.
475
476
32 W. S. Qua, L. Yang, Y. D. Hang, X. Zhang, Y. Qu and J. L. Hua, Sens. Actuators B, 2015,
211, 275.
477
478
33 X. F. Hou, Z. S. Li, B. L. Li, C. H. Liu and Z. H. Xu, Sens. Actuators B, 2018, 260, 295.
34 Y. Tian, B. C. Zhu, W.Yang, J. Jing and X. L. Zhang, Sens. Actuators B, 2018, 262, 345.
479
480
35 H. Fang,N. Wang, L. Xie, P.C. Huang, K. Y. Deng and F. Y. Wu, Sens. Actuators B, 2019,
294, 69.
481
36 L. H. Liu, Q. Zhang, J. Wang, L. L. Zhao, L. X. Liu and Y. Lu, Talanta, 2019, 198, 128.
27

New Journal of Chemistry
Page 28 of 29
1
2
3
4
5
View Article Online
DOI: 10.1039/C9NJ03375K
482
37 X. Y. He, X. F. Wu, W. S. and H. M. Ma, Chem. Commun., 2016, 52, 9410.
6
7
8
9
483
484
38 H. J. Xiang, H. P. Tham, M. D. Nguyen, S. Z. F. Phua, W. Q. Lim, J. G. Liu and Y. L.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Zhao, Chem. Commun., 2017, 53, 5220.
485
486
487
488
39 L. H. Zhai, Z. L. Shi, Y. Y. Tu and S. Z. Pu, Dyes Pigments, 2019, 165, 164.
40 J. Li, Y. Yue, F. Huo and C. Yin, Dyes Pigments, 2019, 164, 335.
41 B. Dong, Y. Lu, N. Zhang, W. Song and W. Zhang, Anal. Chem., 2019, 91, 5513.
42 D. Zhu, X. Yan, A. Ren, W. Xie and Z. Duan, Anal. Chim. Acta, 2019, 1058, 136.
489
490
43 X. Li, H. Ma, J. Qian, T. Cao, Z. Teng, K. Iqbal, W. Qin and H. Guo, Talanta, 2019, 194,
717.
491
492
493
494
495
496
497
498
499
500
44 W. Xuan, Y. Cao, J. Zhou and W. Wang, Chem. Commun., 2013, 18, 10474.
45 K. Huang, M. Liu, Z. Liu, D. Cao, J. Hou and W. Zeng, Dyes Pigments, 2015, 118, 88.
46 L. He, X. Yang, K. Xu and W. Lin, Anal. Chem., 2017, 89, 9567.
47 S. Yang, C. Guo, Y. Li, J. Guo, J. Guo, J. Xiao, Z. Qing, J. Li and R. Yang, ACS Sens.,
2018, 3, 2415,
48 J. Zhang, X. Y. Zhu, X. X. Hu, H. W. Liu, J. Li, L. L. Feng, X. Yin, X. B. Zhang and W. H.
Tan, Anal. Chem., 2016, 88, 11892.
49 K. Huang, L. Yu, P. Xu, X. Zhang and W. Zeng, RSC Adv., 2015, 5, 17797.
50 X. Zhu, M. Xiong, H. Liu, G. Mao, L. Zhou, J. Zhang, X. Hu, X. B. Zhang and W. Tan,
Chem. Commun., 2016, 52, 733.
28

Page 29 of 29
New Journal of Chemistry
1
2
3
4
5
6
7
8
9
View Article Online
DOI: 10.1039/C9NJ03375K
501
502
503
504
51 J. Li, Y. Yue, F. Huo and C. Yin, Dyes Pigments, 2019, 164, 335.
52 B. Dong, Y. Lu, N. Zhang, W. Song and W. Lin, Anal. Chem. 2019, 91, 5513.
53 L. Fan, W. Zhang, X. Wang, W. Dong, Y. Tong, C. Dong and S. Shuang, Analyst, 2019,
144, 439.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
29