Selective Fluorescent Detection of Cysteine over Homocysteine and Glutathione by a Simple and Sensitive Probe

Article abstract of DOI:10.1071/CH17208

A simple fluorescent probe able to selectively and sensitively detect cysteine (Cys) with an excellent dose-dependent relationship between fluorescence intensity and concentration of Cys from 0 to 100M has been designed and synthesised.

Full text of DOI:10.1071/CH17208

CSIRO PUBLISHING
Aust. J. Chem.
Communication
http://dx.doi.org/10.1071/CH17208
Selective Fluorescent Detection of Cysteine over
Homocysteine and Glutathione by a Simple and
Sensitive Probe
A
,
B
A
A
A
Yan-Fei Kang,
Hai-Xia Qiao, Ya-Li Meng, Zhen-Hui Xin,
A
A
A
A
Li-Ping Ge, Ming-Yan Dai, Zhang He, and Cun-Hui Zhang
A
College of Laboratory Medicine, Hebei North University, 11 Diamond Street South,
Zhangjiakou 075000, Hebei Province, China.
B
Corresponding author. Email: kangyanfei172@163.com
A simple fluorescent probe able to selectively and sensitively detect cysteine (Cys) with an excellent dose-dependent
relationship between fluorescence intensity and concentration of Cys from 0 to 100 mM has been designed and synthesised.
Manuscript received: 16 April 2017.
Manuscript accepted: 6 June 2017.
Published online: 27 June 2017.
Biothiols, such as cysteine (Cys) and homocysteine (Hcy), as
well as glutathione (GSH), are abundant in organisms and play
important roles in a variety of physiological processes such as
surfactants, long response times, and have relatively poor selec-
tivity and sensitivity. Thus, the development of a fluorescent
probe is still needed to selectively detect Cys content.
[14]
[
1]
redox homeostasis and cellular functions. Cys serves as the
precursor of GSH and Coenzyme A. Furthermore, it carries out
crucial roles in protein function, metabolism, detoxification, and
Coumarin, as a natural dye, has attracted much attention in the
scientific community. When coumarin is excited by light, intra-
molecular charge transfer (ICT) occurs. The ICT process can be
enhanced by an electron-donating group at the 7-position. In
addition, a substituent group either at the 3- or 4-position also
[
2]
cellular functions. However, an abnormal level of Cys is
associated with many health disorders. For example, low levels
of Cys are considered to be related to hair depigmentation,
oedema, liver damage, muscle and fat loss, weakness, hemato-
[15]
improves the ICT process via resonance and inductive effects.
Therefore, in this work, we designed and synthesised probe 1
(4-methyl-2-oxo-3-phenyl-2H-chromen-7-yl acrylate) by methyl
substitution at the 4-position and phenyl substitution at the
3-position of coumarin. The ICT has been characterised
(Scheme 1). Probe 1 can discriminate Cys from other thiols, even
Hcy and GSH, in a short time. On the other hand, it is worth
mentioning that there is an excellent dose-dependent relationship
between the fluorescence intensity and concentration of Cys from
0 to 100 mM.
[3]
poiesis reduction, leucocyte loss, psoriasis, and skin lesions.
Elevated levels of Cys have been linked to cardiovascular
complications, neurotoxicity, and Parkinson’s and Alzheimer’s
[
4]
disease. Therefore, the rapid and reliable detection of Cys is of
great importance in clinical applications.
Among various detection techniques, fluorescence detection
has attracted much attention on account of its simplicity,
excellent selectivity and sensitivity, and low cost, as well as
simplicity of operation. To date, a great diversity of fluorescence
probes for Cys has been designed and synthesised on the basis of
First, a base-catalysed Kostanecki condensation was used to
synthesise 7-hydroxy-4-methyl-3-phenylcoumarin (2) from
commercial reagents 2,4-dihydroxyacetophenone and phenyl-
[
5]
[6]
different mechanisms, such as Michael addition, cyclisation
[7]
[8]
with aldehyde, conjugate addition–cyclisation, cleavage of
[16]
acetyl chloride. Subsequently, compound 2 was reacted with
acryloyl chloride to obtain 1 in 24 % yield (Scheme 2; for details
and characterisation, see the Supplementary Material).
[
9]
sulfonamide and sulfonate esters,
[
and cleavage of S–S
10]
bonds.
difficult because of their structural similarity.
However, distinguishing Cys from Hcy and GSH is
Based on a
[
11]
Michael addition reaction mechanism, it’s difficult to distin-
guish between Cys, Hcy, and GSH because the nucleophilic
activity of the sulfhydryl group shows almost no difference.
[
12]
ICT off
ICT on
Meanwhile, some probes have been designed and synthesised
that act with photo-induced electron transfer and intramolecular
proton transfer mechanisms. However, they also cannot differ-
Cys
[
13]
O
entiate between Cys, Hcy, and GSH.
few fluorescence probes have been reported to distinguish Cys
Up to now, although a
O
O
O
HO
O
O
[8]
over Hcy and GSH, there are still some limitations in their
practical application. For example, they require the use of
Scheme 1. Intramolecular charge transfer (ICT) process.
Journal compilation � CSIRO 2017
www.publish.csiro.au/journals/ajc

B
Y.-F. Kang et al.
With probe 1 in hand, initially, its UV-vis absorption and
emission spectra were measured in aqueous buffered solution
pH 7.4 phosphate buffered saline (PBS), containing 10 %
was capable of detecting Cys selectively. Subsequently, we
carried out quantitative analysis for Cys and the results showed
there was an excellent dose relationship between fluorescence
(
2
CH CN). Upon the addition of Cys (100 mM), the maximum
intensity and concentration from 0 to 100 mM (R ¼ 0.9983)
3
absorption of probe 1 (10 mM) was observed to undergo a red-
shift from 315 to 380 nm (Fig. S1, Supplementary Material).
Next, we investigated the fluorescence behaviour of probe 1
in the presence and absence of Cys (Fig. 1). A significant turn-on
fluorescence response at 455 nm was observed with the addition
of Cys. However, probe 1 exhibited a negligible fluorescence
intensity in the presence of other thiol-analytes. Thus, probe 1
(Fig. 2a). This outstanding linear range is wider than most
previous reports. The fluorescence detection limit of Cys was
[
17]
ꢁ8
evaluated (3s/m, n ¼ 20)
to 1.6 ꢀ 10 M, which can satisfy
the detection of Cys under physiological conditions (Fig. S2,
Supplementary Material). Lastly, the quantum yield of probe 1
was measured as F ¼ 0.089 and 0.015 in the presence or absence
[
18]
of Cys.
Cl
O
O
O
O
Cl
K CO , CH Cl
K CO /acetone
2
3
HO
O
O
2
3
2
2
O
O
O
HO
OH
1
2
Probe 1
Scheme 2. Synthesis of probe 1.
250
200
150
100
50
0
2
2
1
1
40
00
60
20
Probe 1 ϩ Cys
8
0
0
0
Probe 1 ϩ other
amino acids
4
A
B
C
D
E
F G
H
I
J
K
L
M
N O P Q R S T
Amino acids
400
450
500
550
600
650
Wavelength [nm]
Fig. 3. Fluorescence intensity of probe 1 (10 mM) in the presence of
various analytes in aqueous buffered solution (pH 7.4 PBS, containing
Fig. 1. Fluorescence spectra of probe 1 (10 mM) in aqueous buffered
solution (pH 7.4 PBS, containing 10 % CH CN) with various amino acids
Cys, Hcy, GSH, N-acetyl-Cys, Phe, Ala, Glu, Lys, Pro, Trp, Thr, Val, Ile,
1
0 % CH
3
CN), lex ¼ 380 nm. Grey bar: probe 1 þ analytes; black bar: probe
3
1
þ Cys þ analytes; A: blank; B: Hcy; C: GSH; D: N-acetyl-Cys; E: Phe;
(
F: Ala; G: Glu; H: Lys; I: Pro; J: Trp; K: Thr; L: Val; M: Ile; N: Asp; O: Ser;
P: Gly; Q: DL-Met; R: Leu; S: L-Met; T: His, 100 mM. Each experiment was
performed in triplicate.
Asp, Ser, Gly, DL-Met, Leu, L-Met, His, 100 mM) and incubation for 6 min at
room temperature, lex ¼ 380 nm. Each experiment was performed in
triplicate.
(a)
(b)
2
2
1
1
50 y ϭ 2.4423x ϩ 2.3061
400
300
200
100
0
240
200
160
120
R2 ϭ 0.9983
00
50
00
probe 1 ϩ Cys
probe 1
probe 1ϩ GSH
probe 1ϩ Hcy
5
0
0
0
20
40
60
80 100
Concentration [µM]
8
0
0
0
4
400
450
500
550
600
0
5
10
15
20
25
30
35
40
Wavelength [nm]
Time [min]
Fig. 2. (a) Emission spectra of probe 1 (10 mM) with increasing concentration of Cys (from 0 to 10 equiv.) in aqueous buffered solution (pH 7.4 PBS,
containing 10 % CH
CN), lex ¼ 380 nm. Inset: dose-dependent relationship between fluorescence intensity and concentration of Cys. (b) Time-dependent
fluorescence intensity of probe 1 (10 mM) in the presence of Cys, Hcy, and GSH (100 mM). Each experiment was performed in triplicate.
3

Coumarin-Based Fluorescent Detection of Cysteine
C
To investigate the interaction between probe 1 and Cys, Hcy,
and GSH, the time-dependent fluorescence intensity response
was measured (Fig. 2b). Probe 1 exhibited insignificant fluores-
cent intensity which did not change as the time increased. Thus,
this result suggests probe 1 is stable in aqueous buffered solution
respectively (Fig. S3, Supplementary Material). A difference of
two orders of magnitude for the reaction rates leads to probe 1
selectively detecting Cys.
Encouraged by these results, we further evaluated the influ-
ence of other amino acids on the selective detection of probe 1
towards Cys. As shown in Fig. 3, a 38-fold enhancement in
fluorescence intensity was quantified for Cys compared with the
starting probe fluorescence. Markedly, when other amino acids
were added, the fluorescence intensity of probe 1 for Cys had no
obvious change. Notably, GSH and Hcy also did not induce a
change in the fluorescence intensity of probe 1 for Cys. Thus,
this result showed that probe 1 could detect Cys selectively
under biological conditions. The effect of pH on fluorescence
intensity was also explored (Fig. S4, Supplementary Material).
First, probe 1 exhibited a very weak fluorescence intensity
which showed little change over pH 2–9. However, with the
(pH 7.4 PBS, containing 10 % CH CN). Moreover, the fluores-
3
cent intensity increased gradually with the increase of reaction
time towards Cys and approached a plateau within 25 min,
[
14d-f]
which is faster than in previous reports.
However, with
the increase of reaction time towards Hcy and GSH, although the
fluorescent intensities were increased, this enhancement is far
lower than for Cys. Meanwhile, we estimated the pseudo-first-
0
order rate constant (k ) in aqueous buffered solution (pH 7.4
PBS, containing 10 % CH CN) at room temperature. The
3
pseudo-first-order rate constants for Cys, Hcy, and GSH were
ꢁ1
measured as k ¼ 1.4 ꢀ 10ꢁ2, 4.0 ꢀ 10ꢁ4, and 3.0 ꢀ 10ꢁ4
0
s ,
O
O
Cys
S
O
O
O
O
O
O
HOOC
Probe 1
NH2
O
NH
+
COOH
S
HO
O
O
2
C16H O ϩ H: 253.0859
12
3
Scheme 3. Proposed reaction mechanism between probe 1 and Cys.
(a)
(b)
(c)
(d)
Fig. 4. Fluorescence images of probe 1. (a) Hela cells. (b) Cells were incubated with DMSO for 1 h. (c) Hela cells were
incubated with probe 1 (10 mM) for 1 h. (d) Hela cells were incubated with probe 1 (10 mM) for 1 h after pre-incubation with
NEM (1 mM) for 30 min. lex ¼ 380 nm, scale bar ¼ 40 mm.

D
Y.-F. Kang et al.
addition of Cys, the fluorescence intensity showed evident
enhancement over pH 7–9. Therefore, probe 1 can be used for
the detection of Cys in the physiological pH range.
Zhangjiakou City (No. 1621122H), and Guide Projects of Department of
Education of Hebei Province (No. Z2017026).
Toexplorethereactionmechanism, weanalysedthemixtureof
probe 1 and Cys by high-resolution mass spectrometry (HRMS)
References
[1] X. Chen, Y. Zhou, X. Peng, J. Yoon, Chem. Soc. Rev. 2010, 39, 2120.
doi:10.1039/B925092A
[2] (a) K. G. Reddie, K. S. Carroll, Curr. Opin. Chem. Biol. 2008, 12, 746.
doi:10.1016/J.CBPA.2008.07.028
þ
whereby a peak at m/z 258.0856 (compound 2, [Mþ H] ) was
clearly identified (Fig. S5, Supplementary Material). Moreover,
we analysed the mixture of probe 1 and Cys by HPLC, the results
of which also evidenced formation of compound 2 from the
reaction between probe 1 and Cys (for analysis method, see the
Supplementary Material). Based on the above a probable reaction
mechanism between probe 1 and Cys is given in Scheme 3. First,
conjugation of Cys to the a,b-unsaturated carbonyl moiety and
subsequent intramolecular cyclisation produced compound 2
which leads to the remarkable fluorescence turn-on response of
probe 1 in aqueous buffered solution (pH 7.4 PBS, containing
(
b) C. E. Paulsen, K. S. Carroll, Chem. Rev. 2013, 113, 4633.
doi:10.1021/CR300163E
[
3] (a) S. Shahrokhian, Anal. Chem. 2001, 73, 5972. doi:10.1021/
AC010541M
(
b) H. P. Wu, C. C. Huang, T. L. Cheng, W. L. Tseng, Talanta 2008, 76,
3
47. doi:10.1016/J.TALANTA.2008.03.004
4] (a) H. Refsum, P. M. Ueland, O. Nygard, S. E. Vollset, Annu. Rev.
[
Med. 1998, 49, 31. doi:10.1146/ANNUREV.MED.49.1.31
(b) J. G. Klingman, D. W. Choi, Neurology 1989, 39, 397.
(c) M. T. Heafield, S. Fearn, G. B. Steventon, R. H. Waring, A. C.
[8]
1
0% CH CN).
3
Williams, S. G. Sturman, Neurosci. Lett. 1990, 110, 216. doi:10.1016/
Finally, to explore the practical application of probe 1, we
incubated probe 1 with different concentrations of calf serum at
78C. The quantitative results indicated that the fluorescence
intensity increased with an increase of calf serum content
Fig. S6, Supplementary Material). In addition, we investigated
0
304-3940(90)90814-P
(
d) B. K. Rani, S. A. John, Biosens. Bioelectron. 2016, 83, 237.
3
doi:10.1016/J.BIOS.2016.04.013
5] (a) L.-Y. Niu, Y.-Z. Chen, H.-R. Zheng, L.-Z. Wu, C.-H. Tung, Q.-Z.
[
(
Yang, Chem. Soc. Rev. 2015, 44, 6143. doi:10.1039/C5CS00152H
the application of using probe 1 to determine the presence of
Cys. The cytotoxic activity of probe 1 was measured in Hela
cells by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
(
b) H. S. Jung, X. Chen, J. S. Kim, J. Yoon, Chem. Soc. Rev. 2013, 42,
019. doi:10.1039/C3CS60024F
6
(c) Y. Zhou, J. Yoon, Chem. Soc. Rev. 2012, 41, 52. doi:10.1039/
C1CS15159B
[19]
trazolium bromide) method
before bioimaging. Results
showed that probe 1 (20 mM) didn’t induce obvious cell death
in 24 h (Fig. S7, Supplementary Material). Therefore, probe 1
[6] (a) N. Sok, M. Nikolantonaki, S. Guyot, T. D. Nguyen, A.-S. Viaux,
F. Bagala, Y. Rousselin, F. Husson, R. Gougeon, R. Saurel, Sens.
Actuators B 2017, 242, 865. doi:10.1016/J.SNB.2016.09.171
(10 mM) can be safely used in cell systems in 1 h. Subsequently,
(
b) T. Liu, F. Huo, J. Li, J. Chao, Y. Zhang, C. Yin, Sens. Actuators B
016, 232, 619. doi:10.1016/J.SNB.2016.04.014
c) T. Liu, F. Huo, C. Yin, J. Li, J. Chao, Y. Zhang, Dyes Pigm. 2016,
28, 209. doi:10.1016/J.DYEPIG.2015.12.031
d) Z. Chen, Q. Sun, Y. Yao, X. Fan, W. Zhang, J. Qian, Biosens.
we investigated the imaging of probe 1 in HeLa cells (Fig. 4).
When Hela cells were incubated with probe 1 (10 mM) for 1 h,
the cells presented moderately blue fluorescence. However,
with pre-incubation of N-ethyl maleimide (NEM, 1 mM) for
2
(
1
(
3
0 min, the blue fluorescence didn’t occur. Thus, probe 1 is
Bioelectron. 2017, 91, 553. doi:10.1016/J.BIOS.2017.01.013
[7] (a) H. Y. Lee, Y. P. Choi, S. Kim, T. Yoon, Z. Guo, S. Lee, K. M. K.
Swamy, G. Kim, J. Y. Lee, I. Shin, J. Yoon, Chem. Commun. 2014, 50,
6967. doi:10.1039/C4CC00243A
capable of detecting Cys in living Hela cells by biological
imaging.
In conclusion, in this work, we have designed and synthesised
probe 1 on the basis of substituents at the 3/4-position of
coumarin. Interestingly, probe 1 displays a remarkable fluores-
cence enhancement over a physiological pH range upon the
addition of Cys. This provides the necessary conditions for
the probe to be applied to cell systems. There is only a single
methene difference between Cys and Hcy in their molecular
structures, which confers difficulty for selective detection of Cys.
However, results evidenced that probe 1 possesses satisfactory
selectivity and sensitivity for detection of Cys. Meanwhile, there
is an excellent dose-dependent relationship between fluorescence
intensity and concentration of Cys from 0 to 100 mM which
provides the conditions for quantitative detection of Cys in a
practical application. In addition, the fluorescence intensity of
probe 1 responded well to the calf serum. More importantly, its
lower detection limit and bioimaging potential make probe 1 a
perfect candidate for application in biological systems.
(
b) C. Chen, W. Liu, C. Xu, W. Liu, Biosens. Bioelectron. 2016, 85, 46.
doi:10.1016/J.BIOS.2016.04.098
[
8] (a) J. Wang, B. Li, W. Zhao, X. Zhang, X. Luo, M. E. Corkins, S. L.
Cole, C. Wang, Y. Xiao, X. Bi, Y. Pang, C. A. McElroy, A. J. Bird,
Y. Dong, ACS Sens. 2016, 1, 882. doi:10.1021/ACSSENSORS.5B00271
(
b) C. Y. Kim, H. J. Kang, S. J. Chung, H.-K. Kim, S.-Y. Na, H.-J. Kim,
Anal. Chem. 2016, 88, 7178. doi:10.1021/ACS.ANALCHEM.
B01346
6
(c) Z.-H. Fu, X. Han, Y. Shao, J. Fang, Z.-H. Zhang, Y.-W. Wang,
Y. Peng, Anal. Chem. 2017, 89, 1937. doi:10.1021/ACS.ANA
LCHEM.6B04431
(
d) X. Dai, X. Kong, W. Lin, Dyes Pigm. 2017, 142, 306. doi:10.1016/
J.DYEPIG.2017.03.045
e) Y.-J. Fu, Z. Li, C.-Y. Li, Y.-F. Li, P. Wu, Z.-H. Wen, Dyes Pigm.
017, 139, 381. doi:10.1016/J.DYEPIG.2016.12.033
(
2
[
9] (a) Q. Gao, W. Zhang, B. Song, R. Zhang, W. Guo, J. Yuan, Anal.
Chem. 2017, 89, 4517. doi:10.1021/ACS.ANALCHEM.6B04925
(
b) C. Yin, W. Zhang, T. Liu, J. Chao, F. Huo, Sens. Actuators B 2017,
46, 988. doi:10.1016/J.SNB.2017.02.176
(c) X. Dai, T. Zhang, J.-Y. Miao, B.-X. Zhao, Sens. Actuators B 2016,
23, 274. doi:10.1016/J.SNB.2015.09.106
d) W. Zhang, C. Yin, Y. Zhang, J. Chao, F. Huo, Sens. Actuators B
016, 233, 307. doi:10.1016/J.SNB.2016.04.089
2
Supplementary Material
2
Synthesis and characterisation of probe 1 and other experi-
mental details are available on the Journal’s website.
(
2
[
10] D.-P. Li, J.-F. Zhang, J. Cui, X.-F. Ma, J.-T. Liu, J.-Y. Miao, B.-X.
Zhao, Sens. Actuators B 2016, 234, 231. doi:10.1016/J.SNB.2016.
04.164
Acknowledgements
This work was supported by the Natural Science Foundation of Hebei
Province (No. B2016405026), Young Elitist Foundation of Hebei Province
[11] N. Shao, J. Y. Jin, S. M. Cheung, R. H. Yang, W. H. Chan, T. Mo,
(No. BJ2016003), Guidance Projects of Science and Technology of
Angew. Chem. Int. Ed. 2006, 45, 4944. doi:10.1002/ANIE.200600112

Coumarin-Based Fluorescent Detection of Cysteine
E
[
[
[
12] (a) L. Long, L. Zhou, L. Wang, S. Meng, A. Gong, F. Du, C. Zhang,
[15] X. Liu, J. M. Cole, P. G. Waddell, T.-C. Lin, J. Radia, A. Zeidler,
J. Phys. Chem. A 2012, 116, 727. doi:10.1021/JP209925Y
[16] (a) S. Neelakantan, P. V. Raman, A. Tinabaye, Indian J. Chem. 1982,
21B, 256.
(b) L. Xie, Y. Takeuchi, L. M. Cosentino, A. T. McPhail, K.-H. Lee,
J. Med. Chem. 2001, 44, 664. doi:10.1021/JM000070G
[17] (a) D. MacDougall, W. B. Crummett, Anal. Chem. 1980, 52, 2242.
doi:10.1021/AC50064A004
Org. Biomol. Chem. 2013, 11, 8214. doi:10.1039/C3OB41741G
(b) S. Madhu, R. Gonnade, M. Ravikanth, J. Org. Chem. 2013, 78,
5056. doi:10.1021/JO4004597
13] (a) K.-H. Hong, D. I. Kim, H. Kwon, H.-J. Kim, RSC Adv. 2014, 4, 978.
doi:10.1039/C3RA42935K
(b) M. Lan, J. Wu, W. Liu, H. Zhang, W. Zhang, X. Zhuang, P. Wang,
Sens. Actuators B 2011, 156, 332. doi:10.1016/J.SNB.2011.04.042
14] (a) X. Ren, F. Wang, J. Lv, T. Wei, W. Zhang, Y. Wang, X. Chem,
Dyes Pigm. 2016, 129, 156. doi:10.1016/J.DYEPIG.2016.02.027
(b) K.-H. Hong, S.-Y. Lim, M.-Y. Lun, J.-W. Lim, J.-H. Woo,
H. Kwon, H.-J. Kim, Tetrahedron Lett. 2013, 54, 3003. doi:10.1016/
J.TETLET.2013.03.119
(c) Y.-F. Kang, H.-X. Qiao, Y.-L. Meng, S.-J. Cui, Y.-J. Han, Z.-Y.
Wu, J. Wu, X.-H. Jia, X.-L. Zhang, M.-Y. Dai, RSC Adv. 2016, 6,
94866. doi:10.1039/C6RA19267J
(
b) D. P. Murale, H. Kim, W. S. Choi, D. G. Churchill, RSC Adv. 2014,
, 5289. doi:10.1039/C3RA47280A
c) L. Wang, Q. Zhou, B. Zhu, L. Yan, Z. Ma, B. Du, X. Zhang, Dyes
Pigm. 2012, 95, 275. doi:10.1016/J.DYEPIG.2012.05.006
d) X. Dai, Q.-H. Wu, P.-C. Wang, J. Tian, Y. Xu, S.-Q. Wang, J.-Y.
4
(
(
[18] (a) A. A. Musse, J. Wang, G. P. deLeon, G. A. Prentice, E. London,
A. R. Merrill, J. Biol. Chem. 2006, 281, 885. doi:10.1074/JBC.
M511140200
Miao, B.-X. Zhao, Biosens. Bioelectron. 2014, 59, 35. doi:10.1016/
J.BIOS.2014.03.018
(
e) P. Das, A. K. Mandal, G. U. Reddy, M. Baidya, S. K. Ghosh,
(b) J. J. Jankowski, D. J. Kieber, K. Mopper, P. J. Neale, Photochem.
Photobiol. 2000, 71, 431. doi:10.1562/0031-8655(2000)071,0431:
DAIOUS.2.0.CO;2
A. Das, Org. Biomol. Chem. 2013, 11, 6604. doi:10.1039/
C3OB41284A
(f) S. Q. Wang, Q. H. Wu, H. Y. Wang, X. X. Zheng, S. L. Shen, Y. R.
[19] R. F. Hussain, A. M. E. Nouri, R. T. D. Oliver, J. Immunol. Methods
Zhang, J. Y. Miao, B. X. Zhao, Analyst 2013, 138, 7169. doi:10.1039/
1993, 160, 89. doi:10.1016/0022-1759(93)90012-V
C3AN01440A