A curcumin-analogous fluorescent sensor for cysteine detection with a bilateral-response click-like mechanism

Article abstract of DOI:10.1016/j.saa.2020.118879

A novel curcumin-analogous fluorescent sensor, DNP, was developed for cysteine detection with a bilateral-response click-like mechanism. DNP indicated high selectivity and practical sensitivity. It could recognize Cys from other biologically relevant molecules, especially, from GSH and Hcy. The most interesting point was that, with typical azide groups for sensing, DNP indicated a covalent binding procedure with Cys instead of a presupposed simple reduction for reductive sulfide. Moreover, the recognition occurred at both sides of the sensor. DNP could be utilized into the detection of endogenous and exogenous Cys in living cells. Though the specific optical performances of DNP still need optimization, this work supplied novel information for broadening the vision on fluorophores and mechanisms, for the monitoring of Cys and even other sulfur-containing species.

Full text of DOI:10.1016/j.saa.2020.118879

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 245 (2021) 118879
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A curcumin-analogous fluorescent sensor for cysteine detection with a
bilateral-response click-like mechanism
b,
a,b,
Fang Fang a, Sheng-Jin Liu a, Xiang-Jun Fan c, , Yu-shun Yang , Zhen Li
⁎
⁎
⁎
a
College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China
b
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences; State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University,
No.163 Xianlin Road, Nanjing 210023, China
c
Affiliated Hospital of Nantong University, Nantong 226001, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 June 2020
Received in revised form 20 August 2020
Accepted 21 August 2020
Available online 27 August 2020
A novel curcumin-analogous fluorescent sensor, DNP, was developed for cysteine detection with a bilateral-
response click-like mechanism. DNP indicated high selectivity and practical sensitivity. It could recognize Cys
from other biologically relevant molecules, especially, from GSH and Hcy. The most interesting point was that,
with typical azide groups for sensing, DNP indicated a covalent binding procedure with Cys instead of a
presupposed simple reduction for reductive sulfide. Moreover, the recognition occurred at both sides of the sen-
sor. DNP could be utilized into the detection of endogenous and exogenous Cys in living cells. Though the specific
optical performances of DNP still need optimization, this work supplied novel information for broadening the vi-
sion on fluorophores and mechanisms, for the monitoring of Cys and even other sulfur-containing species.
© 2020 Elsevier B.V. All rights reserved.
Keywords:
Cysteine detection
Fluorescent sensor
Curcumin analogue
Bilateral response
Click-like mechanism
1. Introduction
pre-clinical diagnose approaches [22,23]. Among them, according to the
research heat, Cys acts as the pioneering target.
Following the investigations of nitro oxide [1] and carbon monoxide
[2] as possible gasotransmitters with their reported roles in physiology
and pathological processes, the study on the sulfur-containing signaling
molecules has become a hotspot [3–6]. Given the significances in both
composition and metabolism, biological mercaptans seemed the core
group in the sulfur-related internal circulation and balance [7–9]. The
most common mercaptans in organisms are cysteine (Cys) [10], homo-
cysteine (Hcy) [11] and glutathione (GSH) [12]. The variation of their
physiological concentrations can offer reliable basis for the diagnosis
of certain diseases or disorders [13]. Taken Cys as a representative, ex-
cessive Cys can act as the indices for cardiovascular diseases, motor neu-
ron disease, Parkinson's Disease and Alzheimer's Disease [14–17], while
insufficient Cys can be the possible cause of liver damage, hair discolor-
ation, edema, growth retardation, and muscle relaxation [18–21]. Thus,
the unique detection of the biological mercaptans is of great significance
for revealing the corresponding physiological procedures and proposing
Existing approaches for Cys detection contained mass spectrometry
[24], fluorescence analysis [25], electrochemical analysis [26], high per-
formance liquid chromatography (HPLC) [27] and capillary electropho-
resis [28]. Particularly, in the field of life science, small molecular
fluorescent sensor, with high sensitivity, simple operation, strong visi-
bility and low toxicity, has become indispensable monitoring imple-
ment [29]. One most urgent challenge is the distinguishing ability
among the three mercaptans, because their chemical structures are
quite similar. More and more thorough study on the reaction mecha-
nism has brought typical recognition groups for Cys, such as acrylate
[30–38], N-ethylmaleimide [39,40], chloracetate ester [41], α,β-
unsaturated ketone [42,43] and nitrobenzoxadiazole [44]. Among the
most recent Cys sensors [45], the selectivity from GSH and Hcy was es-
pecially focused on [46,47]. Still, novel sensing mechanism with unique
cases are in emergency.
In this work, we developed a curcumin-analogous fluorescent sen-
sor, DNP (“D” for double, “N” for azide, “P” for Probe), for cysteine detec-
tion with a bilateral-response click-like mechanism (Scheme 1). Though
the azide group was once reported, the recognition mechanism here
was not a presupposed simple reduction. Interestingly, the sensor
DNP reacted with Cys through a covalent binding strategy instead.
⁎
Corresponding authors.
E-mail addresses: fxj7245@sina.com (X.-J. Fan), ys_yang@nju.edu.cn (Y. Yang),
lizhenzfx@nju.edu.cn (Z. Li).
https://doi.org/10.1016/j.saa.2020.118879
1386-1425/© 2020 Elsevier B.V. All rights reserved.

2
F. Fang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 245 (2021) 118879
Scheme 1. Schematic illustration of selective detection for Cys by the sensor DNP.
With the sensing procedures occurring in both sides, DNP indicated
high selectivity and practical sensitivity. The sensor also suggested
good applicability in living cells, offering novel information for
exploiting the feasibility of tracking Cys in real applications.
2.3. Synthesis of sensor DNP
The synthetic route to prepare the sensor DNP was generally shown
in Scheme 2. Consulting our previous work [48], we adjusted the substi-
tutes with actual demands. Detailed synthesis procedures of DNP and
the intermediates were as follows.
2. Experimental
3. Results and discussion
2.1. Materials and general methods
3.1. Synthesis of DNP
All chemicals were purchased from commercial suppliers and used
without further purification. Chromatographic purification was per-
formed with 200–300 mesh silica gel. 1H NMR and 13C NMR spectra
were recorded by using Bruker DRX-400. Mass spectra were carried
out on AB SCIEX Triple TOF 4600 mass spectrometer (U.S.A) by Mass
Spectrometry Facility at Nanjing University. The infrared spectra were
received by Fourier transform infrared spectrometer NEXUS870. The
pH measurements were conducted on pH meter of PHS-25. All UV–vis
spectra were tested on Shimadzu UV-2550 spectrometer and
fluorescence intensity was tested on Hitachi F-7000 Fluorescence Spec-
trophotometer. The imaging experiments were performed by using
two-photon confocal fluorescent microscope (Leica TCS SP8 MP, Nan-
jing University). The excitation wavelength in cell imaging experiments
was set as 395 nm.
All the solvents were of analytic grade. The stock solutions of all the
tested anions were prepared from NaAc, NaCl, Na2CO3, NaHSO3 Na2S,
NaF, Na2HPO4, NaNO3, NaSCN, Na2SO4, Na2S2O3 with doubly distilled
water. The stock solution of amino acids was prepared in doubly dis-
tilled water. Stock solution of DNP (10 mM) was prepared in DMSO,
which was diluted to the required concentrations for measurement.
The samples were excited at 395 nm with the excitation and slit widths
both set at 5.0 nm. The photomultiplier voltage was set at 650 V. The
emission spectra were scanned from 410 nm to 700 nm at 1200 nm/
min.
The sensor DNP was synthesized as described in Scheme 2. and con-
firmed by satisfactory spectroscopic data (1H NMR, 13C NMR, HRMS and
IR, Figs. S3–12, SI). The sensing mechanism might be inferred from the
HRMS spectra of DNP and the detecting product DNP-Prod
(Figs. S3–12, SI).
3.2. General fluorescent properties and sensitivity for Cys
Fluorescence and absorption properties of the sensor DNP (5 μM)
were tested in PBS buffer (10 mM, 0.5% DMSO, pH 7.4). As shown in
Fig. 1a, the free sensor DNP showed a major absorption peak at
400 nm. When Cys (60 eq) was added, the maximum absorption spec-
trum exhibited a 2-fold increased at 400 nm, whereas decreased at
450–650 nm and the color of the solution faded. Meanwhile, in
Fig. 1b, the sensor DNP itself had negligible fluorescence, however,
after Cys (60 eq) was added, a large fluorescence enhancement at
510 nm could be observed, accompanied by the phenomenon that the
solution exhibited bright green fluorescence.
To evaluate the sensitivity of DNP for Cys, we then investigated the
change of fluorescence spectral via varying the concentrations of Cys
(1–60 eq). As depicted in Fig. 2, with continuous increasing concentra-
tions of Cys, the fluorescence intensity at 510 nm of the system gradu-
ally increased (Fig. 2a). The plateau was at 35 eq of Cys, with an
approximately 62-fold fluorescent enhancement compared with the
free sensor. Moreover, an excellent linear relationship (R2 = 0.9976)
between the fluorescence intensity at 510 nm and the Cys concentra-
tions in the range of 0–5.0 eq was also acquired (Fig. 2b). The detection
limit of DNP for Cys was then calculated as 0.37 μM according to the
slope of the standard curve, which was not in top class but barely prac-
tical for a novel case. These results indicated that DNP could monitor Cys
qualitatively before being further optimized. The time-dependent fluo-
rescence changes of DNP towards Cys were also investigated. As
2.2. Ethics statement
All experiments were conducted in accordance with the guideline is-
sued by the National Medical Products Administration (NMPA of China).
Cell lines were cultured and sub-cultured in accordance with the guide-
lines established by the National Science Council of Republic China.

F. Fang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 245 (2021) 118879
3
Scheme 2. The synthesis route of the sensor DNP.
depicted in Fig. 2c & d, when fixing the concentration of Cys, the sensing
process could reach the saturation within 120 min. No obvious further
enhancement at 510 nm could be observed with the extension of re-
sponse time. When the time-dependent stability of the detecting sys-
tem was evaluated, the result indicated that the system could be
stable even after a 48 h incubation (Fig. S1, SI). Additionally, the pH-
dependent stability was also checked (Fig. S2, SI). In the range from
3.0 to 11.0, with the increase of pH, the fluorescence intensity at
510 nm increased and reaching the plateau at 7.0, then tended to be sta-
ble up to 11.0. DNP itself showed negligible fluorescent signal under all
the tested pH conditions. Thus, DNP exhibited a relatively wide range in
suiting pH conditions.
fluorescence changes caused by other amino acids and ions were all
very limited comparing with that caused by Cys. These results indicated
that DNP was high selective for Cys and could be further utilized for
fluorescence detection of Cys in physiological conditions.
3.4. Proposed mechanism
The more familiar the recognition group and the fluorescent evalua-
tion procedures were, the larger pleasantly surprise there actually was
when we looked into the recognition mechanism in this unique case.
Empirically, we had presupposed the mechanism to be a simple reduc-
tion, thus reductive sulfide might be the major detecting target. How-
ever, several clues denied the original presumption. The most evident
hint was that DNP exhibited high selectivity towards Cys instead of
other typical reductive sulfide. The other key evidence was that the fluo-
rescent wavelength of the detecting product indicated a blue shift to the
green region compared with the presupposed orange region. Mean-
while, we also had to check whether DNP would experience a regiose-
lective response to react at only one side as previously reported [48].
Initially, the Job's plot analysis was carried out to confirm the number
of reacted sites on DNP when treated with Cys. As shown in Fig. 4a,
with the fixed total concentration of 10 μM, when the reciprocal shift oc-
curred in the relative concentrations of DNP and Cys, the fluorescence
intensity of the system reflected corresponding variation. As a result,
the fluorescence intensity reached the peak at the ratio of [Cys]/
[DNP] = 2:1, demonstrating that the stoichiometry in this case was a
3.3. Selectivity of DNP towards Cys
In order to evaluate the selectivity of DNP towards Cys over various
relevant molecules, we examined the fluorescence of DNP in response
to various amino acids (Ala, Arg, Asn, Asp, Gln, Glu, Gly, Hcy, His, Ile,
Leu, Lys, Met, Pro, Phe, Ser, Trp, Thr, Val, Tyr,), possible interfering ions
(Ac−, Cl−, CO32−, F−, HPO24−, HSO−3 , NO3−, SCN−, S2O32−, SO42−, S2−
)
and thiols (GSH, Hcy). As exhibited in Fig. 3a, DNP showed high selectiv-
ity for Cys over other analytes. Specially, no significant fluorescence re-
sponse of DNP could be observed to the relevant molecules such as GSH
and Hcy. To further demonstrate the ability of monitoring Cys in the
presence of other competitive molecules, the anti-interference perfor-
mance of DNP was also investigated. As shown in Fig. 3b, the
Fig. 1. (a) UV–visible spectral response of DNP (5 μM) in the absence and presence of Cys (60 eq) in PBS buffer (10 mM, pH 7.4, 0.5% DMSO) at 37 °C for 2 h; (b) Fluorescence emission
spectra of DNP (5 μM) in the absence and presence of Cys (60 eq) in PBS buffer (10 mM, pH 7.4, 0.5% DMSO) at 37 °C for 2 h. λEx: 395 nm, λEm: 510 nm. Excitation and emission slit
widths = 5.0 nm, voltage = 650 V. The data represent the average of three independent experiments. The inset showed the photographs of DNP in the absence and presence of Cys
under visible light or a UV 365 nm lamp.

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F. Fang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 245 (2021) 118879
Fig. 2. (a) The fluorescence spectra of DNP (5 μM) with various concentrations of Cys in PBS buffer (10 mM, pH 7.4, 0.5% DMSO) at 37 °C for 2 h. (b) The nonlinear (0–60 eq) and linear
(0–5.0 eq) relationship between the fluorescence intensity at 510 nm of DNP (5 μM) and the concentrations of Cys in PBS buffer (10 mM, pH 7.4, 0.5% DMSO). (c) The fluorescence spectra
of DNP (5 μM) with Cys (60 eq) in different time periods (0–480 min). (d) The fluorescence intensity of DNP (5 μM) with Cys (60 eq) in different time periods (0–480 min). Excitation
wavelength = 395 nm, excitation and emission slit widths = 5.0 nm, voltage = 650 V. The data represent the average of three independent experiments.
Fig. 3. (a) The fluorescence intensity at 510 nm of DNP (5 μM) incubated with various reagent in PBS buffer (10 mM, pH 7.4, 0.5% DMSO) at 37 °C for 2 h. 1) Control, 2) Ac−, 3) Ala, 4) Arg,
5) Asn, 6) Asp, 7) Cl−, 8) CO32−, 9) F−, 10) Gln, 11) Glu, 12) Gly, 13) Hcy, 14) His, 15) HPO42−, 16) HSO3−, 17) Ile, 18) Leu, 19) Lys, 20) NO3−, 21) Met, 22) Pro, 23) Phe, 24) SCN−, 25) S2O23−
,
26) SO24−, 27) Ser, 28) Thr, 29) Trp, 30) Val, 31) Tyr, 32) S2−, 33) GSH, 34) Cys. Cys: 300 μM. Other: 600 μM. (b) The fluorescence intensity at 510 nm of DNP (5 μM) incubated with Cys
(60 eq) and various reagent in PBS buffer (10 mM, pH 7.4, 0.5% DMSO) at 37 °C for 2 h. 1) Ala (600 μM), 2) Arg (600 μM), 3) Asn (600 μM), 4) Gln (600 μM), 5) Glu (600 μM), 6) Gly
(600 μM), 7) His (600 μM), 8) Ile (600 μM), 9) Leu (600 μM), 10) Lys (600 μM), 11) Met (600 μM), 12) Phe (600 μM), 13) Pro (600 μM), 14) Ser (600 μM), 15) Thr (600 μM), 16) Trp
(600 μM), 17) Tyr (600 μM), 18) Val (600 μM), 19) Ac− (600 μM), 20) Cl− (600 μM), 21) CO23− (600 μM), 22) F− (600 μM), 23) HPO42− (600 μM), 24) NO−3 (600 μM); 25) SCN−
(600 μM); 26) SO24− (600 μM); 27) S2O−3 (600 μM); 28) Cys (300 μM). Excitation wavelength = 395 nm, emission wavelength = 510 nm, excitation and emission slit widths =
5.0 nm, voltage = 650 V. The data represent the average of three independent experiments.

F. Fang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 245 (2021) 118879
5
Fig. 4. (a) Job's plot analysis of DNP with Cys. The total concentration of DNP and Cys was 10 μM in PBS buffer (10 mM, pH 7.4, 0.5% DMSO). The reaction was conduction at 37 °C for 2 h.
(b) The comparison between HRMS spectra of DNP and the detecting product DNP-Prod. (c) Mechanism diagram of the reaction between DNP and Cys.
bilateral-response type. As the corresponding macroscopic phenome-
non, the bilateral-response fluorescence was stronger than that of an re-
gioselective response. Therefore the key point was what on earth the
detecting product was. The comparison between HRMS spectra of
DNP and the detecting product DNP-Prod was displayed in Fig. 4b.
The HRMS of free probe DNP (ESI-TOF m/z): [C19H13BF2N6O2 + Na]+
was calculated as 429.1059, and from the mass spectrometry we
found 429.1057. After the DNP reacting with Cys, we found 615.1954
([C25H27BF2N4O6S2 + Na]+) from the mass spectrometry. According
to these results, we could sketch the contours of the recognition mech-
anism. As illustrated in Fig. 4c, the detecting procedure experienced a
click-like reaction with the nitrogen molecules separating, therefore re-
sulted in the corresponding product DNP-Prod. As a newly-reported
mechanism, we could only attribute the selectivity to the experimental
data, however, we supposed that the selectivity of DNP was more close
to the first stage of acrylate-recognition procedure, which picks Cys
from GSH or Hcy via the nucleophilic ability [45–47].
30 min, extra incubation with Cys for 120 min could still cause a
dose-dependent fluorescent enhancement as shown in Fig. 5g–l. With
positive correlation between the fluorescence intensity and the Cys con-
centration, it became possible to quantify Cys with the measured fluo-
rescence signal. Meanwhile, DNP was low-toxic or even non-toxic to
the cultured cells under the experimental conditions at the concentra-
tion of 10 μM according to the cell morphology.
4. Conclusion
In conclusion, a novel curcumin-analogous fluorescent sensor, DNP,
was developed for cysteine detection with a bilateral-response click-
like mechanism. As the most interesting point of this unique case, the
recognition group azide experienced a covalent binding procedure
with Cys instead of a presupposed simple reduction. The recognition oc-
curred at both sides of the sensor. As a preliminarily trial, DNP indicated
high selectivity and practical sensitivity. Especially, it could pick Cys
from other various biologically relevant species, including two similar
biological thiols, GSH and Hcy. It also could achieve the detection of en-
dogenous and exogenous Cys in living HepG2 cells. Though the specific
optical performances of DNP still need further improvement, it supplied
novel information for exploiting the feasibility with a broader vision on
fluorophores and mechanisms.
3.5. Biological imaging
Moreover, we evaluated the capability of the sensor DNP to selec-
tively monitor Cys in living cells (Fig. 5). In the control group, HepG2
cells (human hepatocellular carcinoma cell line) were directly incu-
bated with DNP for 30 min. As shown in Fig. 5a–c, DNP reacted with
the original intracellular Cys thus the fluorescence in the green channel
could be observed. If pretreated with the thiol-blocking reagent NEM
(N-Ethylmaleimide) for 60 min, a further incubation with DNP for
30 min led to much weaker fluorescence as presented in Fig. 5d-f. Fur-
thermore, after being pretreated with NEM for 60 min and DNP for
CRediT authorship contribution statement
Fang Fang: Methodology, Formal analysis. Sheng-Jin Liu: Valida-
tion, Visualization. Xiang-Jun Fan: Writing - review & editing. Yu-

6
F. Fang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 245 (2021) 118879
Fig. 5. Confocal microscopy images of Cys detection in living HepG2 cells using 10 μM DNP. (a–c) HepG2 cells incubated with DNP for 30 min at 37 °C; (d–f) HepG2 cells pretreated with
1 mM NEM for 60 min and subsequently incubated with DNP for 30 min at 37 °C; (g–i) HepG2 cells treated in the same method of (d–f) and then incubated with 300 μM Cys for 120 min at
37 °C; (j–l) HepG2 cells treated in the same method of (d–f) and subsequently treated with 600 μM Cys for 120 min at 37 °C. (m) Quantification of imaging data is presented as means SD
from three independent experiments. **P < 0.01, ***P < 0.001. Excitation wavelength = 395 nm, signals collected in Green channel.
[8] A. Pastore, A. Alisi, G. di Giovamberardino, A. Crudele, S. Ceccarelli, N. Panera, C.
shun Yang: Writing - original draft. Zhen Li: Project administration,
Funding acquisition.
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Declaration of competing interest
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[11] P.A. Outinen, S.K. Sood, S.I. Pfeifer, S. Pamidi, T.J. Podor, J. Li, J.I. Weitz, R.C. Austin,
The authors declare no conflict of interest.
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specific changes in gene expression in human vascular endothelial cells, Blood 94
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