Nanomolar detection of Hcy, GSH and Cys in aqueous solution, test paper and living cells

Article abstract of DOI:10.1039/c4ra13262a

A novel naphthalimide-based fluorescent probe was designed and synthesized for thiol recognition with high sensitivity and excellent selectivity. The probe can detect thiol quantitatively in a concentration range of 0-6.0 μM and the detection limit could

Full text of DOI:10.1039/c4ra13262a

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PAPER
Nanomolar detection of Hcy, GSH and Cys in
aqueous solution, test paper and living cells†
Cite this: RSC Adv., 2015, 5, 4941
a
a
a
a
Xingjiang Liu,‡ab Wenying Zhang,‡ Chunxiao Li, Wan Zhou, Zhanxian Li,
*
a
Mingming Yu and Liuhe Weia
*
A novel naphthalimide-based fluorescent probe was designed and synthesized for thiol recognition with
high sensitivity and excellent selectivity. The probe can detect thiol quantitatively in a concentration
range of 0–6.0 mM and the detection limit could be as low as 1.6 nM. The fluorescence enhancement of
this probe upon addition of Cys on test paper and the application of the probe for selective detection of
intracellular Hcy, GSH and Cys have been successfully demonstrated.
Received 27th October 2014
Accepted 8th December 2014
DOI: 10.1039/c4ra13262a
www.rsc.org/advances
emission spectra are within the visible spectral region. Moreover,
their photophysical properties can be easily tuned through
judicious structural modications.15 In fact, based on 1,8-naph-
Introduction
Biological thiols are essential for maintaining the appropriate
redox status of proteins, cells, and organisms.1 For example, as
an essential amino acid, cysteine (Cys) is involved in protein
synthesis, detoxication, and metabolism.2 Cys deciency is
considered to result in many health problems such as slowed
growth rate, hair depigmentation, edema, lethargy, liver
damage, and so on.3 Similarly, as an important endogenous
antioxidant, glutathione (GSH) is considered to be an indicator
of oxidative stress.4 Therefore, signicant effort has been paid to
the development of colorimetric, phosphorescent, and uores-
cent probes for these thiol-containing amino acids to achieve
high sensitivity, low cost, and ease of detection.5
Most of the developed Cys probes are based on chemical
reactions such as cyclization reactions with aldehydes,6 Michael
additions,7 and cleavage reactions.8 As an especially effective
strategy, thiol-induced cleavage of intramolecular uorescence
quenching agents, such as 2,4-dinitro-benzenesulfonamide or
2,4-dinitro-bensensulfonate, has been demonstrated with great
success using a number of prototypical uorescent chromo-
phores such as uorescein,9 BODIPY,10 resorun,11 rhoda-
mine,12 benzothiazole13 and other species.14
thalimide, some biological thiol-uorescent probes have been
reported.16 However, the reported naphthalimide-based thiol
uorescent sensors are very limited and it is necessary to design
and synthesize novel naphthalimide derivatives for sensing thiol.
Herein we report a 2,4-dinitrobenzenesulfonate derivative of
1,8-naphthalimide, 1 (Scheme 1) that we designed as a uorescent
probe for biological thiols at a physiological value of pH ¼ 7.40
(Fig. 1). Due to a photoinduced electron transfer (PET) process
from the electron donor, uorophore to the electron accepter 2,4-
dinitrobenzenesulfonate moiety, probe 1 is non-uorescent (F ¼
0.031). However, when treated with biological thiols, probe 1
exhibits a relatively rapid, time-dependent enhancement of its
uorescence signal (F ¼ 0.393). Such nding suggests that thiols
selectively cleave the 2,4-dinitrobenzenesulfonate moiety in probe
1, thus eliminating PET-induced uorescence quenching and
concomitantly allowing uorescence to occur (Scheme 1).
Results and discussions
In this work, synthetic route employed for the preparation of
novel 1,8-naphthalimide-based thiol sensitive uorescent probe
As prototypical uorophores, 1,8-naphthalimide-based deriv-
atives have been widely used in uorescence detection and
imaging because they posses many favorable optical properties
such as good photostability, high uorescence quantum yields
and large Stokes' shi and also because their absorption and
aCollege of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou
450001, China. E-mail: lizx@zzu.edu.cn; yumm@zzu.edu.cn; Fax: +86 371
67781205; Tel: +86 371 67781205
bCollege of Chemistry and Chemical Engineering, Central South University, Changsha,
Hunan 410083, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra13262a
Scheme 1 Synthetic route to probe 1 and its reaction with thiols.
RSC Adv., 2015, 5, 4941–4946 | 4941
‡ These authors contributed equally to the work.
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observed upon the addition of Cys (82 fold uorescence
enhancement, F ¼ 0.393), which was ascribed to the prohibi-
tion of PET upon sensing. Fig. 2 indicates the relationship
between the uorescence peak intensity at 521 nm and the
concentration of Cys. In the concentration range of 0 and
3.0 mM, the uorescence peak intensity of 1 is in good linear
relationship with Cys concentration, implying that Cys can be
quantitatively detected in a wider concentration range. From
this linear calibration graph, the detection limit of probe 1 for
Cys is found to be about 1.6 nM based on signal-to-noise ratio
(S/N) ¼ 3,17 which was sufficiently low for the detection of thiols
under physiological conditions. These results led us to conclude
that 1 could be an effective uorescent ‘turn on’ probe for Cys.
The reaction mechanism was studied by 1H NMR and Mass
spectra. The reaction product was isolated by column chroma-
tography over silica gel column to conrm that compound 2 was
formed through the reaction of Cys with probe 1 (Scheme 1). 1H
NMR spectra of the isolated product, the probe 1 itself and the
reference compound 2 are shown in Fig. 3. Upon addition of
Cys, three protons of probe 1 corresponding to the protons of
2,4-dintrobenzenesulfonyl group around 9.06, 8.65 and
8.39 ppm dramatically disappeared. The reaction product was
isolated by column chromatography to conrm that compound 2
was formed through the reaction of Cys with probe 1 (Scheme 1).
For pure probe 1, a characteristic peak at m/z ¼ 578.0958 was
obtained which corresponds to the species [1 + Na]+, whilst on
addition of Cys, the peak at 578.0958 disappeared and a new
peak appeared at m/z ¼ 326.1503 corresponding to the species
[2 + H] (Fig. S5, S6 and S7 in the ESI†).
Fig. 1 Emission spectroscopy of 1 (5.0 ꢀ 10ꢁ6 M, VHEPES buffer :-
V
¼ 7 : 3, pH ¼ 7.4) upon addition of Cys with excitation at 380 nm.
ethanol
Inset: photographs of 1 (5.0 ꢀ 10ꢁ6 M, VHEPES buffer : Vethanol ¼ 7 : 3,
pH ¼ 7.4) before and after addition of 10 equiv. Cys under a UV lamp
(365 nm).
1 is shown in Scheme 1. The reaction of 4-bromo-1,8-naphthalic
anhydride and monoethanolamine gives the product 3 in a
69.2% yield. Compound 2 was obtained with a yield of 73.9% by
the reaction of compound 3 and piperazine. Compound 2 reacts
with 2,4-dinitrobenzenesulfonyl chloride leads to the probe 1 in
a 54.8% yield. The chemical structure of 1 was conrmed by
1
HRMS, FT-IR, H NMR, and 13C NMR.
To investigate the changes in uorescence emission spec-
trascope of 1 upon exposure to Cys, uorescence titrations were
conducted with Cys in aqueous solution of 1 (5.0 ꢀ 10ꢁ6 M,
VHEPES buffer : Vethanol ¼ 7 : 3, v/v, pH ¼ 7.4) (Fig. 1). As is evident
from inspection of Fig. 2, probe 1 exhibits negligible uores-
cence (F ¼ 0.031) in the buffered solution in the absence of Cys
because of the PET process from 1,8-naphthalimide group to
2,4-dintrobenzenesulfonyl unit. However, a signicant turn-on
uorescence response with a maximum at 521 nm was
We also measured the uorescence changes of compound 1
in the presence of Hcy and GSH (Fig. S1 and S2 in ESI†). Upon
addition of Hcy and GSH respectively, similar uorescence
spectra changes of 1 were obtained, indicating the reaction of 1
with Hcy or GSH leads to the same product as that from the
reaction of 1 with Cys. The uorescence peak intensity of 1 is in
good linear relationship with Hcy and GSH concentration (the
concentration range for Hcy is 0–3.0 mM and 0–6.0 mM is for
GSH.) (Fig. S3 and S4 in ESI†), implying that Hcy and GSH can
be quantitatively detected in a certain concentration range.
Fig.
2
Emission intensity of compound
1
(5.0
ꢀ
10ꢁ6 M, Fig. 3 Partial 1H NMR spectra of 1 upon addition of Cys (10.0 equiv.) in
VHEPES buffer : Vethanol ¼ 7 : 3, pH ¼ 7.4) at 521 nm (black dot) as DMSO-d6. (a) Only probe 1, (b) the isolated product after probe 1
a function of addition of Cys water solution; the linearity of peak reacted with Cys in mixture of water and ethanol, (c) the reference
intensity with respect to Cys concentrations (inset).
compound 2.
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Cys, Hcy and GSH were also done. As shown in Fig. 4, the
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reaction of 1 and Cys is completely accomplished within
135 min and Cys can be detected within 3 min when the
concentration of Cys is more or equal to 5 ꢀ 10ꢁ5 M. However,
the reaction rates of compound 1 with Hcy and GSH are much
slower compared to Cys, which may be ascribed to the larger
steric hindrance of Hcy and Gsh. Such result implied that
compound 1 can detect Cys according to the reaction time.
The pH value of solution was found to be essential to the
cleavage reaction. To investigate the pH effect, the uorescence
intensities of 5.0 mM probe 1 at 521 nm in the absence and
presence of 50 mM Hcy, GSH and Cys were examined at pH
range from 1.0 to 14.0. As shown in Fig. 5, the probe 1 itself does
not show uorescence emission from pH 1.0 to 14.0. Upon the
addition of Cys, Hcy and GSH respectively, there was signicant
uorescence increase in pH range of 5.0–10. In the lower pH
range from 1.0 to 4, no uorescence enhancement could be
observed, which might be ascribed to the difficulty for thiols to
cleave the sulfonyl group under acidic condition. On the
contrary, higher pH environment (>10.0) could cause other
reactions which made against the formation of compound 2.
The selectivity of compound 1 was testied in the presence of
other physiologically relevant amino acids (e.g., Asp, Ala, Val,
Phe, His, Leu, Ser, IIe, Trp, Lys, Arg, Pro, Gly, Met, Tyr, Glu, Thr)
at identical conditions. As shown in Fig. 6, 7 and red bars of
Fig. 8, Probe 1 uniquely responds to thiol-containing amino
acids (Hcy, GSH, Cys) to produce a prominent uorescence
enhancement with a maximum at 521 nm, whereas other amino
acids have no inuence. To further assess its utility as a thiol-
selective probe, its uorescence response to Cys in the pres-
ence of typical thiol-free amino acids (green bars of Fig. 8). The
results demonstrated that all of the thioal-free amino acids have
no interference in the detection of Cys. Therefore, it can be
concluded that the probe 1 showed extremely high selectivity
towards thiol at a physiological pH value.
Fig.
5
Fluorescence intensity change of
1
(5.0
ꢀ
10ꢁ6 M,
V
HEPES buffer : Vethanol ¼ 7 : 3) before and after addition of Cys, Hcy and
GSH respectively with different pH.
detection as solid materials for point-of-care detection appli-
cation. Test paper was selected and the uorescence detection
properties were studied by a UV lamp with photography as well
as solid uorescence spectroscope. In the detection process, the
probe spots were prepared by dropping 1 mL 50 mM solutions of
1 Portable ultraviolet lamp. As shown in Fig. 9, the probe spots
emitted bright blue-green uorescence upon the addition of Cys
under UV illumination, while an enhancement of the uores-
cence intensity at 493 nm could be recorded. Additionally, the
probe spots emitted uorescence only upon the addition of
thiols (Fig. S8 in the ESI†), demonstrating the high selectivity of
probe 1 as solid materials toward thiols.
The practical utility of using probe 1 to detect thiols in an
image-wise manner within living SH-SY5Y cells was explored
(Fig. 10). When SH-SY5Y cells were incubated with 5 mM probe 1
at 37 ꢂC for 30 min, a signicant green uorescence inside the
cells was observed with the aid of an inverted uorescence
As shown in the above, probe 1 can detect thiols in aqueous
solution, it inspired us to further investigate the possibility of
Fig. 6 Fluorescence spectra of 1 (5.0 ꢀ 10ꢁ6 M, VHEPES buffer : Vethanol
Fig. 4 Kinetics of fluorescence increase rate at 521 nm by the reaction ¼ 7 : 3, pH ¼ 7.4) upon addition of 10 equiv. of various amino acids
of 1 (5.0 ꢀ 10ꢁ6 M, VHEPES buffer : Vethanol ¼ 7 : 3, pH ¼ 7.4) and Cys (Asp, Ala, Val, Phe, His, Leu, Ser, IIe, Trp, Lys, Arg, Pro, Gly, Met, Tyr, Glu,
(dark)/Hcy (red)/GSH (green) with excitation at 380 nm.
Thr, Hcy, GSH, Cys) with excitation at 380 nm.
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Fig. 7 Photographs of 1 (5.0 ꢀ 10ꢁ6 M, VHEPES buffer : Vethanol ¼ 7 : 3) upon addition of 10 equiv. of various amino acids (Asp, Ala, Val, Phe, His, Leu,
Ser, IIe, Trp, Lys, Arg, Pro, Gly, Met, Tyr, Glu, Thr, Hcy, GSH, Cys) taken under a UV light (365 nm).
microscope. Thus, probe 1 is capable of permeating into cells
All spectral characterizations were carried out in HPLC-grade
and reacting with thiols to give easily discernable uorescent solvents at 20 ꢂC within a 10 mm quartz cell. UV-vis absorption
images. In a controlled experiment, the SH-SY5Y cells were pre- spectroscopy was measured with a TU-1901 double-beam UV-vis
treated with 1 mM of N-ethylmaleimide (NEM), a thiol-blocking
reagent, aer subsequent incubation with probe 1, no intra-
cellular uorescence was observed within the cells. Therefore,
the probe 1 is applicable for thiol detection in living cells.
Experimental section
Instruments
Mass spectra were obtained on high resolution mass spectrometer
(IonSpec4.7 Tesla FTMS-MALDI/DHB). FT-IR spectra were recorded
on a NEXUS-470 spectrometer at frequencies ranging from 400 to
4000 cmꢁ1. Samples were thoroughly mixed with KBr and pressed
into pellet form. 1H and 13C NMR spectra were recorded on a Bruker
400 NMR spectrometer. Chemical shis are reported in parts per
million using tetramethylsilane (TMS) as the internal standard.
Fig. 8 Fluorescence responses of 1 (5.0 ꢀ 10ꢁ6 M, VHEPES buffer : Vethanol
¼
7 : 3, pH ¼ 7.4) upon addition of different physiologically relevant amino
acids (10 equiv. of amino acid relative to 1) (red bars) and fluorescence
changes of the mixture of 1 and Cys after addition of an excess of the
indicated amino acids (20 equiv. relative to 1) (green bars). The excitation
wavelength was 380 nm. I0 represents the emission intensity at 521 nm in
the fluorescence spectroscopy of compound 1. I represents the emission Fig.
9
(a) Images of test papers for the detection of Cys at
intensity at 521 nm in the fluorescence spectroscopy of compound 1 after various concentrations (0, 2.5 ꢀ 10ꢁ4 M, 1.0 ꢀ 10ꢁ3 M, 5.0 ꢀ 10ꢁ3 M,
addition of the amino acids to the solution of 1 (red bars) and of the 5.0 ꢀ 10ꢁ2 M, 0.1 M, from left to right) in PBS solutions (5.0 ꢀ 10ꢁ5 M,
mixture of 1 and Cys after addition of an excess of the amino acids (green pH ¼ 7.4) under a UV lamp (365 nm). (b) Fluorescence spectra and (c)
bars). The amino acids used were Asp, Ala, Val, Phe, His, Leu, Ser, IIe, Trp, fluorescence intensity at 493 nm of the probe spots of 1 (5.0 ꢀ 10ꢁ5 M)
Lys, Arg, Pro, Gly, Met, Tyr, Glu, Thr, Hcy, GSH, Cys.
on test papers upon addition of various concentrations of Cys
(0–9.0 mM). Other conditions are the same as in Fig. 1.
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3098, 2929, 1648, 1582, 1383. 1H NMR (400 MHz, CDCl3, TMS):
dH 8.61 (m, 1H), 8.54 (d, 1H), 8.45 (m, 1H), 7.72 (m, 1H), 7.22
(d, 1H), 4.47 (t, 2H), 3.99 (t, 2H), 3.27 (t, 4H), 3.22 (t, 4H). 13C
NMR (100 MHz, CDCl3): dC 165.44, 165.01, 133.04, 131.46,
130.76, 130.03, 126.13, 125.66, 122.92, 116.17, 114.96, 62.11,
54.43, 46.22, and 42.76.
Synthesis of compound 1
5 mL dichloromethane solution of 2,4-dinitrobenzenesulfonyl
chloride (81.9 mg, 0.31 mmol) was slowly added to the 25 mL
dichloromethane solution of compound 2 (100 mg, 0.31 mmol)
and the mixture was reacted for 1 h at 0 ꢂC. The nal product 1
(0.0943 g, 54.8%) was obtained by column chromatography over
silica gel column using dichloromethane/ethanol (100 : 1) as
eluent.
Characterization of 1. HRMS (EI) m/z: calcd for C24H21N5O9S
[M + Na]+, 578.0958; found, 578.0955. FT-IR (KBr, cmꢁ1), 3441,
3106, 2924, 2849, 1694, 1652, 1589, 1555, 1538, 1383, 1371,
1352, 1235, 1172, 1121, 1026, 951, 784, 753. 1H NMR (400 MHz,
DMSO-d6, TMS): dH 9.06 (s, 1H), 8.65 (d, 1H), 8.39 (m, 4H), 7.76
(m, 1H), 7.34 (d, 1H), 4.80 (s, 1H), 4.10 (t, 2H), 3.60 (t, 6H), 3.35
(t, 2H), and 3.30 (t, 2H). 13C NMR (100 MHz, DMSO-d6): dC
164.02, 163.51, 155.00, 150.73, 148.38, 134.58, 132.86, 132.32,
131.07, 130.63, 129.41, 127.49, 126.73, 125.85, 123.12, 120.57,
117.05, 116.26, 58.28, 52.54, 46.36, and 42.11.
Fig. 10 Confocal fluorescence contrast images of living SH-SY5Y
incubated with 5 mM probe 1 for 30 min at 37 ꢂC (b and d) and pre-
treated with 1 mM NEM, followed by incubation with probe 1 for
30 min at 37 ꢂC (a and c). (a and b) Fluorescence images; (c and d)
bright field images.
Spectrophotometer, and uorescence spectroscopy was deter-
mined on a Hitachi F-4500 spectrometer. The uorescence
ꢂ
quantum yields were measured at 20 C with quinine bisulfate
in 1 M H2SO4 (F ¼ 0.546) selected as the ref. 18. Cells imaging
was performed with an Olympus IX83 inverted microscope.
Synthesis of the product from the reaction of 1 with Cys
Materials and reagents
55.5 mg (0.1 mmol) probe 1 was dissolved in 15 mL methanol
All commercial grade chemicals and solvents were purchased
and were used without further purication. The probe 1 was
synthesized according to Scheme 1.19
and 121 mg (1 mmol) Cys was dissolved in water. The mixture of
ꢂ
the above two solutions were stirred for 20 h at 20 C and the
nal product (compound 2, 19.7 mg, 61.0%) was obtained by
column chromatography over silica gel column using
dichloromethane/ethanol (1 : 1) as eluent.
Synthesis of compound 3
28 mL 4-bromo-1,8-naphthalic anhydride (2.76 g, 10.0 mmol)
ethanol solution was heated to 80 ꢂC under nitrogen atmo-
sphere and 9 mL monoethanolamine was added to the above
solution. Aer the mixture was reuxed for 4 h, the reaction
liquid was cooled to room temperature and ltered. The lter
cake was washed for 3 times with methanol and the product 3
was obtained (2.2137 g, 69.2%).
Characterization of the product from the reaction of 1 with
Cys (2). HRMS (EI) m/z: calcd for C18H20N3O3 [M + H], 326.1505;
1
found, 326.1503. H NMR (400 MHz, DMSO-d6, TMS): dH 8.44
(t, 2H), 8.38 (d, 1H), 7.79 (t, 1H), 7.30 (d, 1H), 4.81 (s, 1H), 4.13
(t, 2H), 3.60 (t, 2H), 3.33 (s, 1H), 3.14 (t, 4H), 3.01 (t, 4H).
1
Characterization of 3. H NMR (400 MHz, DMSO-d6, TMS):
Imaging of SH-SY5Y cells
dH 8.50 (m, 2H), 8.27 (m, 1H), 8.16 (m, 1H), 7.96 (m, 1H), 4.82
(s, 1H), 4.13 (t, 2H), 3.63 (t, 2H). 13C NMR (100 MHz, DMSO-d6):
dC 163.44, 163.39, 132.94, 131.94, 131.77, 130.19, 129.44, 129.21,
128.74, 123.31, 122.53, and 58.17.
SH-SY5Y cells (Neuroblastoma cells) were seeded in a 12-well
plate in Dulbecco's modied Eagle's medium (DMEM) supple-
mented with 10% fetal bovine serum and 1% penicillin. The
cells were incubated under an atmosphere of 5% CO2 and 95%
air at 37 ꢂC for 24 h. Before the experiments, cells were washed
with PBS buffered solution three times. The cells were incu-
Synthesis of compound 2
The mixture of 3 (1.4060 g, 4.39 mmol), piperazine (0.3781 g, bated with probe 1 (5 mM) at 37 ꢂC for 30 min. Cell images were
4.39 mmol), and 2-methoxyethanol (35 mL) was reuxed for 5 h. acquired aer washing with PBS buffer three times. For control
The reaction liquid was ltered and the lter cake was obtained experiment, SH-SY5Y cells were pretreated with 1 mM of
ꢂ
as the crude product. The nal product 2 (1.0762 g, 73.9%) was N-ethylmaleimide (NEM) for 30 min at 37 C for 30 min. Aer
obtained by column chromatography over silica gel column washing three times with PBS buffered solution, the pretreated
using dichloromethane/ethanol (1 : 1) as eluent.
cells were incubated with probe 1 (5 mM) at 37 ꢂC for additional
Characterization of 2. HRMS (EI) m/z: calcd for C18H20N3O3 30 min. Fluorescence imaging was performed aer washing the
[M + H], 326.1505; found, 326.1507. FT-IR (KBr, cmꢁ1), 3424, cells three times with PBS buffered solution.
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Conclusions
In summary, based on selective cleavage of 1 by thiols, a “turn-
on” uorescent probe 1 toward thiols has been developed. In
aqueous solution containing 30% ethanol, probe 1 exhibited
ultrasensitive uorescence enhancement for thiols (the LOD
can be as low as 1.6 nM) and a good selectivity over other amino
acids. The uorescence turn-on responses of 1 on test paper and
for imaging cellular thiols were also applied.
Acknowledgements
We are grateful for the nancial supports from National Natural
Science Foundation of China (50903075 and J1210060), New
Teachers' Joint Fund for Doctor Stations from Ministry of
Education of the People's Republic of China (20114101120003)
and Zhengzhou University.
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