A selective and sensitive fluorescent probe for homocysteine and its application in living cells

Article abstract of DOI:10.1016/j.dyepig.2017.01.047

Biological thiols, such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) are important for maintaining the cellular redox equilibrium and play critical roles in physiological and pathological processes. It is necessary to detect Cys, Hcy and GS

Full text of DOI:10.1016/j.dyepig.2017.01.047

Dyes and Pigments 140 (2017) 212e221
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Dyes and Pigments
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A selective and sensitive fluorescent probe for homocysteine and its
application in living cells
,
, b
Xiaoying Qiu a, Xiaojie Jiao a, Chang Liu a b, Dasheng Zheng a, Kun Huang a
,
Qing Wang a b, Song He a, Liancheng Zhao a b, Xianshun Zeng a
,
,
, b, *
a Tianjin Key Laboratory for Photoelectric Materials and Devices, and Key Laboratory of Display Materials & Photoelectric Devices, Ministry of Education,
School of Materials Science & Engineering, Tianjin University of Technology, Tianjin 300384, China
b School of Materials Science and Engineering, Harbin Institute of Technology, Harbin 150001, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Biological thiols, such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) are important for
maintaining the cellular redox equilibrium and play critical roles in physiological and pathological
processes. It is necessary to detect Cys, Hcy and GSH for obtaining important physiological and patho-
logical information, which would promote the development of the medical profession. In this paper, we
have successfully developed a new fluorescent probe OH1-NBD for Hcy detection. Based on the nucle-
ophilic aromatic substitution (SNAr) and rearrangement cascade reaction and the distance-dependent
PET process, OH1-NBD shows to be a selective turn-on fluorescent probe for Hcy with a fast (a few
minutes) and sensitive (16 nM) response. It can function as a fluorescence probe for imaging of Hcy in the
living cell. At the same time, it can also be used for living cells as a potential probe for biothiol-relevant
oxidant-governed redox status assay. We expect that the probe will provide a valuable approach to gain a
better understanding of the roles of biothiols especially Hcy.
Received 11 December 2016
Received in revised form
18 January 2017
Accepted 19 January 2017
Available online 21 January 2017
Keywords:
Fluorescent probe
Thiol
Homocysteine
7-nitro-1,2,3-benzoxadiazole
Imaging
© 2017 Elsevier Ltd. All rights reserved.
1. Introduction
atherothrombosis and coronary heart disease [4], vascular disease
[5], Alzheimer's disease [6] and ischemic stroke [7]. Thus, facile
In recent years, biologically relevant cellular biothiols, such as
glutathione (GSH), cysteine (Cys) and homocysteine (Hcy), have
been paid much attention for their vital roles in vivo. The levels of
these biologically relevant biothiols in the human body are closely
related to human health. Alternations in the levels of these bio-
thiols are associated with a number of diseased states, such as ac-
quired immune deficiency syndrome (AIDS), cancer, and
cardiovascular diseases etc [1]. Especially, abnormal levels of indi-
vidual biothiols may relevant some diseases. It has been reported
that abnormal GSH levels could induce liver damage, leucocyte loss,
psoriasis and cancers [2]. A sustained deficiency of Cys may cause
various health problems such as retarded growth, hair depigmen-
tation, lethargy, liver damage, and skin lesions [3]. An elevated level
of Hcy is considered to be a significant independent risk factor for
method for the determination of these biologically and medically
relevant biothiols in human plasma is highly desirable and of great
interest.
Some conventional methods for biologically relevant thiols
detection, such as high-performance liquid chromatography
(HPLC), UV/vis absorption spectrophotometry, capillary electro-
phoresis, potentiometry, and mass spectrometry (MS), are not only
expensive and time-consuming in practice, but also need highly
trained individuals [8]. On the contrary, optical detection methods,
especially fluorescence imaging methods, exhibit certain unique-
ness and indispensability for the evaluation of the physiological
processes of these biothiols at the cellular/subcellular level due to
their excellent temporal and spatial resolution capability in living
cells. Thus, many efforts have been recently paid for designing of
fluorescent probes for detection and estimation of these biologi-
cally and medically relevant biothiols and exploiting their appli-
cations in bioimaging [8 g], [9e14]. Moreover, appropriate design of
reaction-based probe by taking advantage of the strong nucleo-
philicity of the sulfhydryl group to trigger the release of fluores-
cence signal from the fluorophore is the main strategy being
* Corresponding author. Tianjin Key Laboratory for Photoelectric Materials and
Devices, and Key Laboratory of Display Materials & Photoelectric Devices, Ministry
of Education, School of Materials Science & Engineering, Tianjin University of
Technology, Tianjin 300384, China.
E-mail address: xshzeng@tjut.edu.cn (X. Zeng).
http://dx.doi.org/10.1016/j.dyepig.2017.01.047
0143-7208/© 2017 Elsevier Ltd. All rights reserved.

X. Qiu et al. / Dyes and Pigments 140 (2017) 212e221
213
resorted to for the design of thiol-specific probes. Based on this
strategy, a number of excellent fluorescent probes by cyclization
reaction with aldehyde [9], Michael addition [10], disulfide bond
cleavage [11], sulfonate ester and sulfonamide cleavage [12], G-
quadruplex switchable strategy [13a] etc, have been designed and
synthesized for identification of biothiols. However, many docu-
mented thiol-selective probes tend to respond to classes of analytes
instead of individual biothiol [13b]. At the same time, many probes
also suffer from some drawbacks, such as long response time
(>30 min), only working in high ratio of organic solvents, and
complex synthesis. Therefore, the design of novel and improved
probes for biothiols is a topic of continued importance.
The 7-nitro-1,2,3-benzoxadiazole (NBD) is a widely used fluo-
rophore for molecular design of various analyte-responsive fluo-
rescent probes. Recently, Pluth et al. reported that 4-substuented
NBD derivatives showed excellent selectivities toward biothiols
via the nucleophilic aromatic substitution (SNAr) reaction of bio-
thiols with the electron-deficient NBD moiety at the 4-C position
[14]. Based on this strategy, a few NBD-based probes for Cys, H2S
and GSH have been successfully developed [15]. Although varieties
of thiol-selective probes have been reported and used with some
success in biological applications, up to date only a few probes
exhibit higher selectivity toward Hcy over other biothiols and
aminoacids [16]. Enlightened by Pluth's work, we reported herein a
novel cell-permeable fluorescent probe OH1-NBD for Hcy by taking
full advantage of the SNAr and rearrangement cascade reaction of
Hcy with the electron-deficient NBD moiety at the 4-C position, as
well as a distance-dependent photoinduced electron transfer (PET)
mechanism (Schemes 1 and 2). OH1-NBD exhibits a selective turn-
on fluorescent detection of Hcy with a fast (within 10 min) and
sensitive (16 nM) response. The probe can function as a fluores-
cence sensor for imaging of Hcy in the living cell. At the same time,
OH1-NBD can also be used as a potential probe for biothiol-relevant
oxidant-governed redox status assay.
absorption spectra were recorded on a UV-2550 UVeVis spectro-
photometer (Shimadzu, Japan). Fluorescence measurements were
performed using an F-4600 fluorescence spectrophotometer
(Hitachi, Japan) and a quartz cell 1 cm. Melting point was recorded
on a Boethius Block apparatus. Live cell fluorescent images were
taken under a FV1000 Confocal Microscope (Olympus).
2.2. The preparation of OH1-NBD
Under argon, compound 1 (1 g, 5.4 mmol) and p-hydroxy
benzaldehyde (0.7 g, 1 mmol) were dissolved in dry acetonitrile
(25 mL). Piperidine (0.0085 g, 0.01 mmol) was added and the so-
lution was stirred at 40 ꢀC till starting material disappeared
(detected by TLC plate). After evaporation of acetonitrile under
pressure, 50 mL of H2O was added to the mixture. The mixture was
extracted with dichloromethane (50 mL ꢁ 3), the combined organic
solution was dried over Na2SO4 and then the solvent was removed
in vacuo. The residue was purified by column chromatography
using dichloromethane to give orange solid OH1. Yield: 1.2 g.
(76.4%); m.p.: 208e210 ꢀC. 1H NMR (400 MHz, DMSO-d6, ppm):
10.00 (s, 1H), 7.56 (d, 8.6 Hz, 2H), 7.24 (d, 16.2Hz, 1H), 7.18 (d,
16.2 Hz,1H), 6.8 (s,1H), 6.8 (d, 3.16 Hz, 2H), 3.4 (s,1H), 2.6 (s,1H),1.0
(s, 1H). 13C NMR (100 MHz, DMSO-d6, ppm): 171.1, 160.3, 157.5,
139.1, 130.8, 128.0, 127.1, 122.3, 116.8, 115.0, 114.2, 75.8, 43.2, 39.1,
32.5, 28.3.
To an ice-bath cooled (0 ꢀC) dry dichloromethane solution (5 ml)
of compound OH1 (174 mg, 0.6 mmol) and 4-chloro-7-
nitrobenzofurazan (NBD) (239 mg 1.2 mmol), DIPEA (200
mL,
1.2 mmol) was added in dropwise. The reaction mixture was stirred
overnight at room temperature. After removal of the solvent, the
residue was purified by silica gel chromatography (DCM: PE ¼ 1:1,
v/v) to afford pure OH1-NBD as an orange solid (250 mg, 93%). m.p.:
255e256 ꢀC. HRMS: m/z [M]þ ¼ 453.1628; Calcd for C25H19N5O4:
453.1437; 1H NMR (400 MHz, DMSO-d6, ppm): 8.66 (d, J ¼ 8.4 Hz,
1H), 7.91 (d, J ¼ 8.4 Hz, 2H), 7.49 (d, J ¼ 16.2 Hz, 1H), 7.46 (d,
J ¼ 8.4 Hz, 2H), 7.36 (d, J ¼ 16.2 Hz, 1H), 6.92 (s, 1H), 6.82 (d,
J ¼ 8.4 Hz, 1H), 2.64 (s, 2H), 2.57 (s, 2H), 1.04 (s, 6H). 13C NMR
(100 MHz, DMSO-d6, ppm): 169.7, 155.1, 153.5, 152.2, 145.2, 144.1,
135.8, 135.0,134.4,130.6,130.2, 129.9,123.1, 121.0,113.7, 112.9,110.8,
77.2, 43.1, 40.5, 39.2, 32.5, 28.3.
2. Experimental
2.1. Materials and equipments
All cations in the form of nitrate salts, all anions in the form of
sodium salts were purchased from Sigma-Aldrich Chemical Com-
pany and used without further purification. N-ethylmaleimide
(NEM) was purchased from Heowns Chemical Company. Cysteine
(Cys), homocysteine (Hcy), glutathione (GSH), N-acetyl cysteine
(NAC) were purchased from Sigma-Aldrich Chemical Company. All
other chemicals used were local products of analytical grade. All
solvents (analytical grade and spectroscopic grade) were obtained
commercially and used as received unless otherwise mentioned.
NMR spectra were recorded on a Bruker spectrometer at 400 (1H
2.3. Cell culture
L929 cells, a cell line from mouse C3H/An connective tissue,
were cultured in RPMI 1640 medium with 10% FBS at 37 ꢀC in a 5%
CO2 atmosphere. RAW 264.7 cells were cultured in RPMI-1640
medium supplemented with 10% FBS, 100 U/mL penicillin,
100
mg/mL streptomycin, 1 mmol/L sodium pyruvate. Cells were
exponentially cultured as monolayer up to 70e80% confluence.
After optimal growth, cells were detached using trypsin-EDTA (1ꢁ).
All the cell culture work has been performed under sterile
condition.
NMR) MHz and 100 (13C NMR) MHz. Chemical shifts (
d values) were
reported in ppm down field from internal Me4Si (1H and 13C NMR).
High-resolution mass spectra (HRMS) were acquired on an Agilent
6510 Q-TOF LC/MS instrument (Agilent Technologies, Palo Alto, CA)
equipped with an electrospray ionization (ESI) source. UV
2.4. Confocal microscopy imaging
Cells were seeded on 24 well chambered cover glass at a density
of 1 ꢁ 106 cells mLꢂ1 for 24 h. Probes dissolved in DMSO were
added to the cells medium (500 mL) at a varying final concentra-
tions according to the need. After incubating for 30 min, excess
probes were removed by gentle rinsing with phosphate buffered
saline (PBS, pH 7.4) three times. In the comparative experiment, the
cells were pretreated with NEM (N-ethylmaleimide, a thiol-reactive
compound) (300
biothiols. After washing with PBS, the cells were further incubated
with the probe OH1-NBD (0.5
M) in PBS at 37 ꢀC for 30 min. In
m
M) at 37 ꢀC for 30 min to eliminate endogenous
Scheme 1. Synthesis of the probe OH1-NBD. Reagents and conditions: a) benzalde-
hyde, piperidine, CH3CN, 40 ꢀC, 12 h; b) NBD, DIPEA, CH2Cl2, r. t., 12 h.
m

214
X. Qiu et al. / Dyes and Pigments 140 (2017) 212e221
Scheme 2. Proposed response mechanism of OH1-NBD to thiols.
mimic immune systems, the cells were treated with lipopolysac-
charide (LPS; 1 g/mL) for 30 min and phorbol-12-myristate 13-
acetate (PMA; 2.5 g/mL) for 30 min. The media was removed,
and then the cells were washed three times with PBS buffer (pH
7.4). The cells were imaged on Olympus FV1000 confocal laser-
scanning microscope, the excitation filter is 488 nm and its emis-
sion was collected in the range of 516e582 nm or 523e598 nm.
emission curve, and h is the refractive index of the solvent used.
m
Subscripts 1 and B refer to the unknown and to the standard,
respectively. Absorbance of sample and reference at their respec-
tive excitation wavelengths was controlled to be lower than 0.05.
The fluorescence intensity was measured simultaneously at
lex1 ¼ 440 nm and lexB ¼ 500 nm in ethanol.
m
3. Results and discussion
2.5. Determination of the fluorescence quantum yield
3.1. The synthesis of the probe OH1-NBD
Fluorescence quantum yield (F1) was determined by using
rhodamine B (FB ¼ 0.89, in ethanol) as the fluorescence standard.
The quantum yield was calculated using the following equation.
Procedures for the synthesis of the probe OH1-NBD were shown
in Scheme 1. Firstly, 2-(3,5,5-trimethylcyclohex-2-enylidene)
malononitrile 1 [17] was obtained as white solid in 86% yield by the
reaction of isophorone with malononitrile in dry ethanol in the
presence of piperidine. Then, the reaction of 1 with p-hydrox-
ybenzaldehyde afforded the key intermediate OH1 [17] as orange
solid in 76% yield. Finally, the probe OH1-NBD was synthesized as
an orange solid at a yield of 93% by the reaction of OH1 with 4-
AB ꢁ F1 ꢁ lexB
A1 ꢁ FB ꢁ lex1
ꢁ
ꢁ
h1
hB
F1
¼
FB
ꢁ
where F1 is the fluorescence quantum yield, A is the absorbance at
the excitation wavelength, F is the area under the corrected

X. Qiu et al. / Dyes and Pigments 140 (2017) 212e221
215
chloro-7-nitrobenzofurazan in dry DCM in the presence of DIPEA.
The chemical structures of OH1 and OH1-NBD were confirmed by
1H, 13C NMR (Figs. S1eS4).
Hcy acts as an electron donor to partially quench the fluorescence
of NBD via a photoinduced electron transfer (PET) mechanism [18],
the fluorescence intensity of N-NBD-Cys and N-NBD-Hcy are
quenched in different degrees by the distance-dependent PET
processes [19]. Compared with the fluorescence of N-NBD-Cys and
N-NBD-Hcy with high-brightness at 543 nm, the fluorophore OH1
with low-brightness produced by all thiols only shows weak fluo-
rescence at 580 nm. Thus, the strong fluorescent signals of N-NBD-
Cys and N-NBD-Hcy establish the selective optical signals of the
probe toward Hcy/Cys over other thiols and aminoacids. At the
same time, the distance-dependent PET process allows the probe to
discriminate Hcy from Cys via fluorescent intensity changes.
Then, the electron absorption and fluorescence emission spectra
of OH1-NBD and those in the presence of different concentrations
of thiols (Hcy, Cys, GSH, NAC, and thioglycol) were investigated in
details in HEPES/ethanol ¼ 1:2 (10 mM, pH ¼ 7.4, v/v) (Fig. 2 and
Figs. S6eS7). The free probe OH1-NBD showed a strong absorption
band centered at 400 nm. Upon the continuous addition of Hcy/Cys,
the absorption bands at 400 nm decreased gradually, along with
new developed absorption bands centered at 443 nm (Fig. 2a and
c), indicative formations of N-NBD-Cys and N-NBD-Hcy due to their
largely red-shifted absorption properties [14b]. In the cases of GSH,
NAC, and thioglycol (Fig. 2e, Figs. S6a and S7a), however, newly
developed absorption bands centered at 424 nm were founded,
suggestive the formation of NBD thioethers due to their minor red-
shifted absorption properties [14b]. At the same time, the probe
showed significantly enhanced and obviously blue shifted fluo-
rescence signals during the Cys and Hcy titrations due to forma-
3.2. Spectroscopic properties of OH1-NBD toward biothiols
Fig. 1 displayed the histograms of fluorescence emission in-
tensities at 543 nm of the probe OH1-NBD (5
various cations, thiols and amino acids. As shown in Inset of Fig. 1,
the probe OH1-NBD (5 M) exhibited essentially very weak fluo-
mM) in the presence of
m
rescence at 570 nm in aqueous solution, which could be assigned to
the fluorescence emission of the ester derivative of fluorophore
OH1. Upon the addition of 10 equiv. of Hcy and Cys, significantly
enhanced and obviously blue-shifted fluorescence emissions at
543 nm (over 35-fold enhancement for Hcy and 24-fold for Cys)
were emerged and developed, which were ascribed to the Hcy/Cys-
induced nucleophilic aromatic substitution and rearrangement
cascade reaction [14]. In stark contrast, S2- and other thiols, such as
GSH, N-acetyl cysteine (NAC) and thioglycol, generated only weak
emission at 580 nm, which were indicative of the release of free
OH1. The reaction was further confirmed by mass spectral analysis
(Fig. S5). At the same time, no significant changes of OH1-NBD in
emission intensity at 543 nm were elicited by the addition of some
biologically important metal cations, such as Naþ, Kþ, Ca2þ, Mg2þ
,
Cu2þ, and Fe3þ, and other amino acids, such as Phe, Ala, Met, Glu,
Arg, Lys, Tyr, Leu, Trp, Ser, Thr, His, Val, etc. The results indicated
that the probe OH1-NBD can function as a fluorescent probe for
selective detection of Hcy/Cys. Furthermore, the obviously higher
fluorescence enhancement in the presence of Hcy than that of Cys
means that the probe tend to selectively response to Hcy over Cys.
The selective fluorescence signals of the probe OH1-NBD toward
Hcy/Cys over other thiols and aminoacids can be rationally
explained by the nucleophilic aromatic substitution (SNAr) reaction
promoted by the sulfhydryl group of thiols (Scheme 2). As can be
seen from Scheme 2, in the first step, all thiols can react with the
probe to produce a nonfluorescent NBD thioether (such as S-NBD-
tions of high quantum yield products N-NBD-Cys (
F
¼ 0.263) and
N-NBD-Hcy (
F
¼ 0.376), respectively. However, due to very low
quantum yields of the NBD thioethers formed from GSH, NAC, and
thioglycol, they showed a gradual fluorescence enhancement of
OH1 (F
¼ 0.0106) at 580 nm with the increasing concentrations of
these thiols (Fig. 2f, Figs. S6 and S7). As shown in Table 1, the
dissociation constants [20] and the detection limits [21] were ob-
tained from the fluorescence titrations (Fig. 2b, d, 2f, and Figs. S6b
and S7b, and Figs. S8eS17). As can be seen from Table 1, the
dissociation constants (Kd) with these thiols are down to the 10ꢂ5 M
range, suggestive high reactivities between the probe and thiols.
The lower Kd value of Hcy than that of Cys indicated that Hcy is
more reactive than Cys with the probe, which is facilitate its
application for the imaging of Hcy in living systems. The detection
limits (LOD) are down to the 10ꢂ8 M range, indicative high sensi-
tivities toward these thiols.
GSH,
brightness (
NBD-Cys and S-NBD-Hcy can proceed an intramolecular rear-
rangement to form N-NBD-Cys (
¼ 0.263) and N-NBD-Hcy
0.376) with high-brightness, respectively. Because the
F
¼ 0.0047) and release the fluorophore OH1 with low-
F
¼ 0.0106) at the same time. However, thioethers S-
F
(F
¼
electron-riched sulfhydryl group within N-NBD-Cys and N-NBD-
To validate the selectivity of the probe in practice, various cat-
ions, thiols and amino acids were used to evaluate the selectivity of
OH1-NBD toward Hcy by means of fluorescence spectra (Fig. 3). In
the competition experiments, OH1-NBD (5
with 10 equiv of Al3þ, Ca2þ, Cys, Fe3þ, Kþ, Mg2þ, Naþ, Phe, Ala, Met,
Glu, Arg, Lys, Tyr, Leu, Trp, Ser, Thr, His, cystine, Val, GSH, Hcy, S2-
mM) was first mixed
,
HQ, NAC, thiophenol and thioglycol, followed by adding 10 equiv of
Hcy. As can be seen from Fig. 3, in the presence of Al3þ, Ca2þ, Cys,
Fe3þ, Kþ, Mg2þ, Naþ, Phe, Ala, Met, Glu, Arg, Lys, Tyr, Leu, Trp, Ser,
Thr, His, cystine, Val, the emission spectra were almost identical to
that obtained in the presence of Hcy alone. In the cases of the
species containing sulfhydryl group, such as GSH, NAC, thiophenol,
and thioglycol, as well as S2-, the emission intensities diminished to
a different extent to that obtained in the presence of Hcy alone for
the competitive SNAr reaction of the probe with them to produce
portions of nonfluorescent NBD thioethers, but they still have
sufficient turn-on ratio for the detection of Hcy. The competition
results indicated that OH1-NBD can be used as a selective fluo-
rescent probe for the detection of Hcy in the presence of a wide
range of biologically relevant competing species.
Fig. 1. Selective responses of OH1-NBD to Cys/Hcy. Histograms represent the fluo-
rescence enhancements of OH1-NBD (5
m
M) at 543 nm in HEPES: ethanol ¼ 1:2
(10 mM, pH ¼ 7.4, v/v) upon addition of 10 equiv. of cations, thiol and various amino
acids (1: Al3þ, 2: Ca2þ, 3: Cu2þ, 4: NAC, 5: Fe3þ, 6: GSH, 7: Hcy, 8: Cys, 9: HQ, 10: Kþ, 11:
Mg2þ, 12: Naþ, 13: S2-, 14: Phe, 15: thiophenol, 16: Ala, 17: Met, 18: Glu, 19: Arg, 20: Lys,
21: Tyr, 22: Leu, 23: Trp, 24: Ser, 25: Thr, 26: His, 27: Val, 28: cystine, 29: thioglycol, 30:
Blank). Inset: fluorescence spectra of probe OH1-NBD in the presence of different
species (1e29). lex ¼ 440 nm.

216
X. Qiu et al. / Dyes and Pigments 140 (2017) 212e221
Fig. 2. UVevis absorption and fluorescence titration spectra of OH1-NBD (5
spectra of the probe (5 M) with an incremental amount of Hcy; b) Fluorescent titration spectra of the probe (5
lex ¼ 440 nm. Slit: 5.0 nm, 5.0 nm); c) UVevis absorption spectra of the probe (5 M) with an incremental amount of Cys; d) Fluorescent titration spectra of the probe (5
M) with an incremental amount of GSH; f) Fluorescent
M) in the presence of different concentrations of GSH (lex ¼ 440 nm. Slit: 5.0 nm, 10.0 nm).
m
M) with Hcy, Cys and GSH in HEPES/ethanol ¼ 1:2 (10 mM, pH ¼ 7.4, v/v). a) UVevis absorption
M) in the presence of different concentrations of Hcy
M) in the
m
m
(
m
m
presence of different concentrations of Cys (lex ¼ 440 nm. Slit: 5.0 nm, 5.0 nm); e) UVevis absorption spectra of the probe (5
m
titration spectra of the probe (5
m
Table 1
Dissociation constants (Kd) and detection limits (LOD) of Hcy, Cys, GSH, NAC and
thioglycol derived from the fluorescence titrations.
Thiols
Dissociation constants (Kd)
LOD (3s/M)
Hcy
Cys
GSH
NAC
9.4 ꢁ 10ꢂ6
2.1 ꢁ 10ꢂ5
1.0 ꢁ 10ꢂ5
1.1 ꢁ 10ꢂ5
2.1 ꢁ 10ꢂ5
M
M
M
M
M
1.6 ꢁ 10ꢂ8
5.1 ꢁ 10ꢂ8
3.4 ꢁ 10ꢂ8
3.2 ꢁ 10ꢂ8
8.2 ꢁ 10ꢂ8
M
M
M
M
M
thioglycol
3.3. Time-dependent responses of OH1-NBD toward biothiols
To further understand the distinct responding fluorescence
properties of the probe toward different thiols, we then carried out
the time-dependent turn-on fluorescence responses of OH1-NBD
Fig. 3. Change ratio (Fi - F0)/(FHcy--F0) of fluorescence intensity (543 nm) of OH1-NBD
(5 mM) upon the addition of 10 equiv. cations and various amino acids and then 10
equiv. Hcy. 1: Al3þ, 2: Ca2þ, 3: Cys, 4: Fe3þ, 5: GSH, 6: Hcy, 7: HQ, 8: Kþ, 9: Mg2þ, 10:
Naþ, 11: NAC, 12: S2-, 13: Phe, 14: Thiophenol, 15: Ala, 16: Met, 17: Glu, 18: Arg, 19:
Cystine, 20: Lys, 21: Tyr, 22: Leu, 23: thioglycol, 24: Trp, 25: Ser, 26: Thr, 27: His, 28:
Val. Data were collected after 30 min incubation. lex ¼ 440 nm. Slit: 5.0 nm, 5.0 nm.
(5 mM) in the presence of 10 equivalents of Hcy, Cys, GSH, NAC,
thiophenol, S2- and thioglycol at room temperature. As shown in
Fig. 4 and Fig. S18 a-f, a weak and stable fluorescence signal at ca.

X. Qiu et al. / Dyes and Pigments 140 (2017) 212e221
217
Fig. 5. Temperature-dependent fluorescence spectra (543 nm) of OH1-NBD (5
mM)
with 10 equiv of Cys and Hcy in HEPES: ethanol ¼ 1:2 (10 mM, pH ¼ 7.4, v/v).
lex ¼ 440 nm. Slit: 5.0 nm, 5.0 nm.
3.5. Temperature-dependent responses of OH1-NBD toward Hcy
and Cys
To further determine whether OH1-NBD could function under
physiological conditions, we then evaluated its response toward
Hcy and Cys as a function of temperature from 25 to 45 ꢀC (Fig. 5).
As shown in Fig. 5, no fluorescence change of the free probe OH1-
NBD was elicited with the increase of temperature. However, the
fluorescence signals in the presence of Hcy/Cys decreased linearly
with the increase of temperature, but they still have sufficient turn-
on fluorescent ratios for the detection of Hcy and Cys. The results
suggested that the probe could work under physiological
conditions.
Fig. 4. Time-dependent fluorescence responses of the probe OH1-NBD (5
addition of various thiols (Hcy, Cys, GSH, thioglycol, thiophenol and NAC) and S2-
(50
lem ¼ 574 nm lex ¼ 440 nm. Slit: 5.0 nm, 5.0 nm.
mM) upon
m
M) in HEPES: ethanol ¼ 1:2 (10 mM, pH ¼ 7.4, v/v). (a) lem ¼ 543 nm, (b)
3.6. The responses of OH1-NBD toward biothiols in blood serum
It is known that there are three main physiological nonprotein
thiols in blood serum, i.e. Hcy, Cys, and GSH [23]. Based on the
excellent properties of OH1-NBD, we then investigated the fluo-
rescence response of the probe in the presence of fetal calf serum
(FBS) to evaluate the applicability of the probe in biological sam-
ples. As can be seen from Fig. 6, the fluorescence intensity of the
probe increased with increasing blood serum levels after incuba-
tion for 30 min. It is note that the fluorescence intensities at 543 nm
and 580 nm increased equally with low level of serum due to the
higher level of GSH than those of Hcy/Cys in blood serum [23]. The
570 nm was observed with the probe alone. Upon addition of thiols,
rapid increased and blue-shifted fluorescence signals at 543 nm for
Hcy and Cys, and obviously enhanced fluorescence signals at
580 nm for GSH were observed within 2 min, respectively. Subse-
quently, the fluorescence intensity underwent a slow increase and
finally reached a plateau within ca. 10 min (Fig. 4a and b). Although
the fluorescence signals increased at 580 nm upon addition of NAC
and thioglycol, they needed a long time to reach a plateau. On the
other hand, no obvious effect on the fluorescence intensity of the
probe was observed upon the addition of thiophenol and S2-. Thus,
the probe showed rapid turn-on fluorescence processes toward
Cys, Hcy and GSH, and over 80% of the maximum attainable signal
could be reached within 4 min.
3.4. The reaction kinetics of OH1-NBD toward Hcy, Cys and GSH
Then, we examined the reaction kinetics of the probe with Hcy,
Cys, and GSH under pseudo-first-order conditions with a large
excess of these thiols (150 equiv.) over the probe OH1-NBD (5 mM)
[22]. The pseudo-first-order rate constants for Cys, Hcy and GSH
were determined to be k' ¼ 7.5 ꢁ 10ꢂ2 sꢂ1 for Cys, k' ¼ 3.3 ꢁ 10ꢂ2
sꢂ1 for Hcy, and k' ¼ 2.7 ꢁ 10ꢂ3 sꢂ1 for GSH, respectively
(Figs. S19eS21), indicated that the reaction rates of these thiols
with the probe are fast. The results revealed that the probe could
achieve a rapid detection of Hcy, which may facilitate its applica-
tion for the imaging of Hcy in living systems.
Fig. 6. The fluorescence spectra of the probe OH1-NBD (5
different extents of fetal calf serum (0, 10, 30, 50, 100%). Data were collected after
30 min incubation. lex ¼ 440 nm. Slit: 5.0 nm, 5.0 nm.
mM) in the presence of

Fig. 7. Confocal microscope images of L929 cells incubated with the probe OH1-NBD (0.5
(516e582 nm) emission filter. A) Fluorescence image of L929 cells pretreated with N-methylmaleimide (NEM, 300
Fluorescence image of NEM-pretreated L929 cells with the probe for 30 min, and then incubated with Hcy (50 M) for 30 min; C) Fluorescence image of NEM-pretreated L929 cells
with the probe for 30 min, and then incubated with Cys (50 M) for 30 min; D) Fluorescence image of NEM-pretreated L929 cells with the probe for 30 min, and then incubated
with GSH (50 M) for 30 min; E) Fluorescence image of NEM-pretreated L929 cells with the probe for 30 min, and then incubated with NAC (50 M) for 30 min; F) Fluorescence
image of L929 cells incubated with H2O2 (100 M) for 30 min, and then incubated with the probe for 30 min.
mM). Cell images were obtained using an excitation wavelength of 488 nm and a band-path
mM) for 30 min and then incubated with the probe for 30 min; B)
m
m
m
m
m

X. Qiu et al. / Dyes and Pigments 140 (2017) 212e221
219
Fig. 8. Confocal microscope images of RAW 264.7 cells with the probe OH1-NBD (2.5
(523e598 nm) emission filter. a) Fluorescence image of RAW 264.7 cells after 30 min incubation with the probe; b) Fluorescence image of RAW 264.7 cells pretreated with
lipopolysaccharide (LPS; 1 g/mL) for 30 min, phorbol-12-myristate 13-acetate (PMA; 2.5 g/mL) for 30 min, and then incubated with the probe for 30 min; c) Fluorescence image of
RAW 264.7 cells incubated with H2O2 (100 M) for 30 min, and then incubated with the probe for 30 min.
mM). Cell images were obtained using an excitation wavelength of 488 nm and a band-path
m
m
m
enhanced fluorescence intensity of the probe means that the probe
reacts smoothly with Hcy, Cys and GSH in FBS, and can be used as a
potential probe for the assay of the level of thiols in blood serum
samples.
in the living cell. However, only weak enhanced fluorescence sig-
nals in the presence of GSH (50.0 M) and NAC (50.0 M) after
m
m
incubation for 30 min were observed (Fig. 7D and E). Compared
with the IOD of the fluorescence imaging in the presence of OH1-
NBD alone, the changes of IOD in the presence of GSH and NAC
under the same conditions could be neglected (Fig. S22). Additional
observations were made in detecting the oxidant-governed redox
status of biothiols in living cells. In recently, Yoon et al. disclosed
that the intracellular GSH level could be largely decreased by the
H2O2-promoted oxidation [22]. Thus, L929 cells were firstly incu-
3.7. Fluorescence imaging of Hcy in living cells
After determining the reaction kinetics of OH1-NBD for turn-on
fluorescent detection of Hcy/Cys, we then performed a series of
comparative experiments to demonstrate the potentially practical
applications of the probe in sensing of biothiols in live cells (Fig. 7).
To scavenge nonprotein thiols in living cells, the L929 cells were
bated with H2O2 (100
with OH1-NBD (0.5
m
M) for 30 min, and subsequently incubated
m
M) for 30 min. As shown in Fig. 7F, the fluo-
rescence signals in the H2O2-pretreated L929 cell were almost
discernable, indicated that the intracellular biothiols could be
eliminated by the H2O2-promoted oxidation. To confirm that the
fluorescence decrease in the living cell wasn't elicited by H2O2-
promoted direct oxidation of the probe OH1-NBD, the fluorescence
spectra of the probe in the presence of H2O2 was measured in
HEPES: ethanol ¼ 1:2 (10 mM, pH ¼ 7.4, v/v). As shown in Fig. S23,
no change of OH1-NBD in emission intensity at 543 nm was
observed by addition of H2O2. Thus, the quenched fluorescence in
living cells should be resulted from the H2O2-promoted oxidation
of biothiols. On the other hand, compared with the results of NEM
scavenged nonprotein thiols in living cells (Fig. 7A), H2O2 showed to
be more efficient to block the sulfhydryl groups within biothiols via
the H2O2-promoted oxidation (Fig. S24). Therefore, the probe can
firstly treated with 300
mM NEM for 30 min before staining. Then,
OH1-NBD (0.5 M) was added and incubated for another 30 min. As
m
shown in Fig. 7A, only weak fluorescence intensities were observed
in cells with an excitation of green light (488 nm), indicated that
the sulfhydryl groups within nonprotein thiols were mainly
blocked after the administration of NEM. On the contrary, after the
addition of Hcy (50.0 mM) and Cys (50.0 mM), and then incubation
for another 30 min, the fluorescence signals in the green channel
(518e582 nm) increased markedly (Fig. 7B and C). Furthermore, the
plots of integrated optical densities (IOD) of the image in the
presence of Hcy is obviously stronger than that in the presence of
Cys (Fig. S22), which is consistent with the results of fluorescence
measurement in solution. Thus, OH1-NBD is cell-permeable and
can be used as a fluorescence probe for imaging of intracellular Hcy

220
X. Qiu et al. / Dyes and Pigments 140 (2017) 212e221
not only selectively detect Hcy over other biothiols in living cells,
but also can be used for living cells as a potential probe for biothiol-
relevant oxidant-governed redox status assay.
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biothiol-relevant oxidant-governed redox status assay of living
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