Development of near-infrared xanthene fluorescence probe for the highly selective and sensitive detection of cysteine

Article abstract of DOI:10.1016/j.dyepig.2019.107563

Abnormal levels of the biological thiol cysteine (Cys) have been shown to be associated with growth retardation, skin lesions, and neurotoxicity in humans. In order to fully elucidate the role of Cys in biological systems, its levels must be monitored thr

Full text of DOI:10.1016/j.dyepig.2019.107563

Accepted Manuscript
Development of near-infrared xanthene fluorescence probe for the highly selective
and sensitive detection of cysteine
Yongquan Wu, Aiping Shi, Hong Zeng, Yuanyan Li, Huifang Li, Xiaoyong Chen, Wai-
Yeung Wong, Xiaolin Fan
PII:
S0143-7208(19)30528-5
DOI:
https://doi.org/10.1016/j.dyepig.2019.107563
Article Number: 107563
Reference:
DYPI 107563
To appear in: Dyes and Pigments
Received Date: 11 March 2019
Revised Date: 13 May 2019
Accepted Date: 14 May 2019
Please cite this article as: Wu Y, Shi A, Zeng H, Li Y, Li H, Chen X, Wong W-Y, Fan X, Development of
near-infrared xanthene fluorescence probe for the highly selective and sensitive detection of cysteine,
Dyes and Pigments (2019), doi: https://doi.org/10.1016/j.dyepig.2019.107563.
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Graphical Abstract
A novel NIR xanthene fluorescence probe showed sensitive and selective response
toward Cys, and was developed to detect endogenous Cys in vivo.

ACCEPTED MANUSCRIPT
Development of Near-Infrared Xanthene Fluorescence Probe for
the Highly Selective and Sensitive Detection of Cysteine
a,b
a
a
a
a
a
Yongquan Wu , Aiping Shi , Hong Zeng , Yuanyan Li , Huifang Li , Xiaoyong Chen ,
b
a
Wai-Yeung Wong *, and Xiaolin Fan *
School of Chemistry and Chemical Engineering
Organo-Pharmaceutical Chemistry of Jiangxi Province, Gannan Normal University, Ganzhou
a
&
Key Laboratory of
3
41000, P. R. China, E-mail: fanxl2013@gnnu.cn; Fax: +86 797 8393 536; Tel: +86 797
8
393 670
b
Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic
University, Hung Hom, Hong Kong, P. R. China, E-mail: wai-yeung.wong@polyu.edu.hk
Abstract: Abnormal levels of the biological thiol cysteine (Cys) have been shown to
be associated with growth retardation, skin lesions, and neurotoxicity in humans. In
order to fully elucidate the role of Cys in biological systems, its levels must be
monitored through in vitro assays as well as in living cells and animals. Herein, we
designed and synthesized a novel near-infrared xanthene fluorescence probe (NOF1)
with an acrylate group as a trigger moiety to detect Cys over homocysteine and
glutathione. The NOF1 probe exhibited a good selectivity and a high sensitivity
toward Cys, with a detection limit of 210 nM. Additionally, NOF1 retained good
sensitivity and selectivity in human plasma, with a good recovery of Cys within the
range 98% to 102%. Importantly, the NOF1 probe was successfully applied to the
fluorescence imaging of Cys in living cells, zebrafish, and mice, showing great
potential for applications involving the detection of Cys both in vitro and in vivo.
Keywords: Cysteine, Xanthene fluorescence dye, Near-infrared, Fluorescence
imaging
1
Introduction
Biological thiols such as cysteine (Cys), homocysteine (Hcy), and glutathione
(GSH) play important roles in complex physiological systems[1,2], particularly in the
maintenance of the appropriate redox status of proteins, cells, and organisms[3]. Cys

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is an essential amino acid for metabolism and detoxification[4], and its abnormal
levels in living systems have been associated with skin lesions, neurotoxicity,
psoriasis, leucocyte loss, liver damage, and Parkinson’s disease[5-9], among other
illnesses. Therefore, the highly selective and sensitive detection of Cys is of great
significance in biochemistry and biomedicine. Currently available Cys detection
methods include capillary electrophoresis[10,11], high-performance liquid
chromatography, and optical detectors[12-14]. However, these methods require
complicated sample preparation procedures or pretreatment and cannot be used for the
in vivo detection of Cys or in living cells. Nevertheless, in order to fully elucidate the
role of Cys in biological systems, Cys levels must be monitored both in vitro and in
vivo in living cells and animals[15,16]. Thus, the development of convenient and
inexpensive methods for the real-time in vivo monitoring of Cys levels is required.
Fluorescence detection has been extensively studied due to its high sensitivity and
selectivity, as well as its simplicity and facile application in biological sample
assays[17-21]. To date, various fluorescent probes have been developed for the
monitoring of Cys levels based on various mechanisms[3,22-27], and probes to
dectect Cys over Hcy and GSH have been reported[28,29]. However, numerous
probes with emission and absorption wavelengths in the ultraviolet or visible region
cannot be applied in vivo imaging due to their shallow penetration depth and animal
background autofluorescence. Near-infrared (NIR) light (650–900 nm) has a lower
energy, deeper tissue penetration, and reduced optical damage to biological samples
compared to visible light, as well as minimum background interference from
autofluorescence[30-33]. Therefore, the development of novel NIR emission probes
for the rapid, highly selective, and sensitive detection of Cys both in vitro and in vivo
is warranted.
Herein, we designed and synthesized a novel NIR emission xanthene fluorescence
probe (NOF1) with acrylate groups acting as the trigger moiety probe that is able to
sensitively and selectively detect Cys (Scheme 1). Furthermore, the NOF1 probe
undergoes a rapid and selective response for Cys over Hcy and GSH with a lower

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limit of detection (LOD) than the available techniques in vitro. Importantly, the NOF1
probe could be applied for the in vivo imaging of Cys in living cells, zebrafish, and
mice due to its NIR emission, indicating a significant potential for biological
applications.
2
2
Experimental sections
.1 Materials and Methods
Materials. All commercial chemicals were purchased from commercial suppliers
and used without further purification. All solvents were purified before use.
-(4-diethylamino-2-hydroxybenzoyl) beozoic acid, cyclohexanone and HClO (70%)
2
4
were purchased from Shanghai Sain Chemical Technology Co., Ltd. Acryloyl
chloride and potassium acetate were purchased from Adamas Regent Co., Ltd.
4
-Hydroxybenzaldehyde was purchased from Aladdin Technology Co., Ltd. Et N was
3
purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd.
Concentrated H SO was purchased from Jiangsu Tong Sheng Chemical Reagent Co.,
2
4
Ltd. Anhydrous CH Cl was purchased from J&K Scentific Ltd. MTT and PBS were
2
2
purchased from Beyotime Biotechnology Co., Ltd (China). RPMI 1640 was
purchased from Thermo Fisher Scientific Co., Ltd. N-ethylmaleimide (NEM) was
also purchased from Adamas Regent Co., Ltd.
1
Methods. HNMR (400 MHz) spectra was obtained on a BrukerDRX-400
spectrometer, with tetramethylsilane (TMS) as an internal standard (0 ppm)
substances. The high resolution mass spectra (HRMS) spectra were measured with
Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS). UV–vis
absorption spectra were recorded on a Shimadzu UV-2007 spectrophotometer.
Fluorescence measurements were carried out on an Edinburgh FS5 fluorescence
spectrometer. Fluorescence imaging of HeLa cells were obtained using Olympus
FV1000 confocal fluorescence microscope (Japan Olympus Optical Co., Ltd).The
absorbance value was measured using a Thermo Varioskan LUX micro-plate reader
(ThermoFisher Scientific) in the MTT assay. In vivo fluorescence imaging was

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performed with an in vivo imaging system (IVScpoe 7550, Shanghai CLINX Science
Instruments Ltd., China).
2
.2 Synthesis details
The synthesis routine of NOF1 and NOF1-Cys are shown at Scheme S1.
Synthesis of compound 1. Compound 1 was synthesized according to previous
literature[34]. To a solution of 4-Hydroxybenzaldehyde (122 mg, 1 mmol) and Et N
3
(
1.4 mL, 10 mmol) in 15 mL of anhydrous CH Cl , acryloyl chloride (180 mg, 2
2 2
mmol) was added dropwise at 0 °C. After stirring at this temperature 90 min, the
mixture was warmed to room temperature and stirred overnight. The solution was
diluted with CH Cl (30 mL), washed with H O (15 mL × 3) and dried over
2
2
2
anhydrous Na SO . The solvent was removed in rotavapor. The crude product was
2
4
purified by silica gel chromatography (PE/EA) to afford 59 mg (34%) of compound
1
1
2
1
1
as colorless oily liquid. H NMR (400 MHz, CDCl ) δ 10.00 (s, 1H), 7.98 – 7.90 (m,
3
H), 7.37 – 7.31 (m, 2H), 6.65 (dd, J = 17.3, 1.1 Hz, 1H), 6.34 (dd, J = 17.3, 10.4 Hz,
1
3
H), 6.08 (dd, J = 10.5, 1.1 Hz, 1H). C NMR (101 MHz, CDCl ) δ 191.08 (s),
3
63.81 (s), 155.31 (s), 134.00 (s), 133.55 (s), 131.24 (s), 127.39 (s), 122.34 (s).
Synthesis of compound 2. Compound 2 was synthesized according to previous
literature[35]. Cyclohexanone (3.3 mL, 32 mmol) was added dropwise to
concentrated H SO4 (50 mL) and cooled down to °C. Then,
-(4-diethylamino-2-hydroxybenzoyl) beozoic acid (5.01 g, 16 m mol) was added in
0
2
2
portions with vigorous stirring. The reaction mixture was heated at 90 °C for 2 h,
cooled down, and poured onto ice (200 g). Perchloric acid (70%; 3.5 mL) was then
added, and the resulting precipitate was filtered off and washed with cold water (200
mL). Compound 2 obtained as a red solid was used for the next step without further
+
+
purification. HRMS (ESI) C H NO [M+H] : calcd, 377.1991; found, 377.2046.
2
4
26
3
Synthesis of NOF1. Into a 50 mL flask were added compound 3 (123 mg, 0.33
mmol), Compound 2 (59.9 mg, 0.35 mmol), potassium acetate (48 mg, 0.7 mmol) and
acetic acid (5 mL).The mixture was heated to 90 °C for 12 h under nitrogen protection,

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and the solvent was removed by the evaporation under the reduced pressure. The
crude product was purified by silica gel chromatography (CH Cl /MeOH=20:1) to
2
2
1
afford 20 mg (11%) of NOF1 as purple solid. H NMR (400 MHz, CD3OD) δ 8.01
dd, J = 6.8, 2.2 Hz, 1H), 7.94 (s, 1H), 7.59 – 7.51 (m, 4H), 7.17 (d, J = 8.6 Hz, 2H),
(
7.08 (dd, J = 6.4, 2.2 Hz, 1H), 7.01 (s, 3H), 6.51 (d, J = 17.4 Hz, 1H), 6.30 (dd, J =
17.3, 10.4 Hz, 1H), 6.00 (d, J = 9.7 Hz, 1H), 3.59 – 3.53 (m, 4H), 2.86 (dt, J = 10.6,
5.5 Hz, 2H), 2.23 (dt, J = 16.6, 5.8 Hz, 2H), 1.72 (d, J = 4.8 Hz, 2H), 1.21 (t, J = 7.1
+
+
Hz, 6H). HRMS (ESI) C H NO [M+H] : calcd, 534.2275; found, 534.2269.
3
4
32
5
Synthesis of NOF1-Cys. This material was prepared according to the reported
literature[31]. 4-hydroxybenzaldehyde (0.10 g, 0.82 mmol) and compound 3 (0.34 g,
0
.90 mmol) were reacted in 15 mL AcOH overnight, and then the work-up process
was performed. NOF1-Cys was received as dark purple solid (0.10 g, 0.20 mmol),
1
yield: 24%. H NMR (400 MHz, MeOD) δ 8.13 (d, J = 24.6 Hz, 2H), 7.69 – 7.62 (m,
2
H), 7.57 (d, J = 8.3 Hz, 2H), 7.17 (s, 4H), 6.92 (d, J = 8.4 Hz, 2H), 3.70 (q, J = 7.1
Hz, 4H), 2.98 (d, J = 6.9 Hz, 2H), 2.57 – 2.37 (m, 2H), 1.90 – 1.79 (m, 2H), 1.32 (t, J
+
+
=
7.0 Hz, 6H). HRMS (ESI) C H NO [M] : calcd, 480.2169; found, 480.2155.
31 30 4
2
.3 UV–vis absorption and fluorescence spectra
Stock solutions of probe NOF1 (2 mM) was prepared in DMSO. Stock solutions of
analyte (1–10 mM) were prepared in distilled water. The stock solutions of analyte
were diluted to desired concentrations with distilled water when needed. For a typical
optical measurements, probe NOF1 and NOF1-Cys were diluted to 10 µM in PBS (5
mM, pH=7.4) with 50% DMSO (v/v), respectively and 2 mL of the resulting solution
was placed in a quartz cell. The amino acids titration of probe NOF1
spectrophotometric determination was carried out in PBS (5 mM, pH=7.4) with 50%
DMSO (v/v). Various amino acids (20 µM) were titrated into a solution of probe
NOF1 (10 µM), respectively. Before UV-vis absorption and photoluminescence
spectra of the samples were measured, the solutions were kept at 37 °C for 10 min.
For luminescence measurements, excitation was provided at 670 nm, and emission
was collected from 685 to 850 nm. UV-visible spectra were recorded on

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ShimadzuUV-2007 spectrometer and emission spectra were recorded on Edinburgh
FS5 spectrometer.
Human plasma (1 mL) was deproteinized using acetonitrile (3 mL) and centrifuging
at 8,000 rpm for 30 min. The supernatant was diluted in PBS buffer (pH =7.4, 5 mM).
The Cys content in the plasma sample was determined using the same procedure
above and the standard calibration curves.
2
.4 Computational Details
These two compound NOF1 and NOF1-Cys were optimized at the density
functional theory (DFT) level using the Becke’s three-parameter hybrid exchange
functional combined with the Lee-Yang-Parr correlation functional B3YLP[36] and
the 6-31G(d,p) basis set[37]. Harmonic vibrational frequencies were calculated
correspondingly at the same level to confirm each point corresponds to a minimum on
the potential energy surface. Time-dependent DFT (TD-DFT) [38] calculations were
performed to get the nature of the excited states based on the optimized structures. All
calculations were performed with Gaussian 09 Rev. D. 01[39].
2
.5 Cell culture and cytotoxicity of NOF1
The HeLa cell lines (human cervical epitheloid carcinoma) were provided by the
Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences). HeLa
cells were grown in culture media (RPMI 1640) at 37 °C under a humidified
atmosphere containing 5% CO for 24 h. Cells were plated on 15 mm glass coverslips
2
and allowed to adhere for 24 h.
The in vitro cytotoxicity was measured using a standard methyl thiazolyl
tetrazolium (MTT) assay in HeLa cell lines. Cells growing in a 96-well flat-bottomed
4
-1
microplate (1×10 cells well ) in complete RPMI 1640 supplemented with 10% FBS
-1
-1
(
100 µL well ) at 37 °C under 5% CO . After 24h, the sample (100 µL well ) was
2
added to the wells of the test group at concentrations of 5, 10, 15, 20，25，30 µM,
-1
respectively. Added RPMI 1640 supplemented with 0.2 % DMSO (100 µL well ) to
control group. The cells incubated for 24 h. Thereafter, combined 20 µL MTT/PBS

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-1
solutions (5 mg mL ) were added to every well and keep incubating at 37 °C under 5%
-1
CO . After 5 h remove solution and added DMSO (150 µL well ) to wells. The
2
quantity of the formazan product formed as measured the amount of OD 490
(absorbance value) of each well referenced at 690 nm (OD 690) is directly
proportional to the number of living cells in the culture. Each experiment was done in
quadruplicate. The relative viability (%) of cell growth related to control wells
containing cell culture medium without NOF1 was calculated by
[
[
OD]expt/[OD]control×100, where [OD]expt is the absorbance of the test sample and
OD]control is the absorbance of control sample.
2
.6 Confocal imaging for living cells
The cells were incubated for 24 h prior to the imaging experiments. The living cells
were stained with 10 µM probe NOF1 for 30 min at 37 °C under 5% CO . For control
2
experiments, the cells were pretreated with thiol trapping reagent, N-ethylmaleimide
(NEM, 100 µM) for 30 min at 37 °C, followed by washing with PBS buffer (2 mL × 3
times), and incubated with probe NOF1 (10 µM) for 30 min at 37 °C under 5% CO2.
Moreover, HeLa cells were pretreated with Cys (100 µM) for 120 min and then
further incubated with NOF1 (10 µM) for 30 min at 37 °C under 5%. The cells were
imaged under an Olympus FV1000 confocal luminescence microscope. For NOF1,
excited at 635 nm, emission was collected by a range 670–770 nm equipped with a
4
0×-oil immersion objective lens.
2
.7 Visualizing Cys in the living zebrafish
4
-day old zebrafishes were grew in E3 embryo media (15 mM NaCl, 0.5 mM KCl,
1
mM MgSO , 1 mM CaCl , 0.15 mM, KH PO , 0.05 mM Na HPO , 0.7 mM
4
2
2
4
2
4
NaHCO , pH 7.5). As the control group, the 4-day-old zebrafish was incubated with
3
probe NOF1 (10 µM) for 30 min in E3 media at 28 °C. Other group, the 4-day-old
zebrafish was pre-incubated with 200 µM NEM for 30 min and then incubated with
1
0 µM NOF1 for 30 min. After washing with E3 media to remove the remaining
NEM, the zebrafish was further incubated with 10 µM of probe NOF1 in E3 media for
0 min at 28 °C. After washing with E3 media, the zebrafish was imaged by
3

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fluorescence microscopy. The zebrafishes were anesthetized by Tricain, and the low
melting point Agarose served as fixing reagent during imaging.
2
.8 Fluorescence imaging in vivo
Animal procedures were in agreement with the guidelines of the Institutional
Animal Care and Use Committee of Gannan Normal University and performed in
accordance with the institutional guidelines for animal handling. In vivo fluorescence
imaging was performed with a modified luminescence in vivo imaging system
(
IVScpoe 7550, Shanghai CLINX Science Instruments Ltd., China). In this system,
two external 0−5 W adjustable CW 635nm lasers (Changchun Laser Optoelectronics
Technology Ltd., China) and an Andor CCD (IKON-M934BV, Andor Technology
Ltd., UK) were used as the excitation sources and the signal collector, respectively.
Firstly, the mice was anaesthetized in advance with 100 µL chloral hydrate (4% wt.
aqueous solution) and placed carefully into living animal imaging system.
Furthermore, the mouse was subcutaneous injected with 50 µL of 25 µM probe NOF1
in saline. NIR fluorescence images of the living mice were performed at different time.
In addition, in vivo fluorescence imaging system, emission at 680–780 nm was
collected with a pass filter (Semrock, INC) under excitation at 635 nm. Images of
fluorescence signal were analyzed with Kodak Molecular Imaging Software.
3
3
Results and Discussion
.1 Design and synthesis
The NOF1 probe was constructed using a xanthene derivative as the NIR
fluorophore and an acrylate moiety as the reaction site. Xanthene derivatives possess
a large molar extinction coefficient, a high fluorescence quantum yield, and good light
stability, among other properties[40,41]. Furthermore, the acrylate moiety has been
shown to act as an efficient reaction site for biothiols through an addition-cyclization
reaction mechanism[42,43], which generally leads to faster reaction kinetics with Cys
than with Hcy or GSH[44-46]. The addition-cyclization reaction leads to the
formation of a NOF1-Cys product with innate fluorescence. Masking of the phenolic
OH with an acrylate group, an efficient electron acceptor, quenches NOF1-Cys

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fluorescence, yet Cys fluorescence is selectively recovered by the removal of the
masking group through addition-cyclization conjugation (Scheme 2). The NOF1
probe was synthesized through the reaction of intermediate compounds 1 and 2
(
Scheme S1). Furthermore, the chemical structure of NOF1 was confirmed by
H-nucelar magnetic resonance (NMR) and high-resolution mass spectroscopy
HR-MS). The detailed synthetic procedures and structure characterization methods
1
(
were provided in the Experimental Section and Supporting Information (see Figures
S8-S14).
3
.2 UV-vis absorption and fluorescence spectra
The absorption and emission spectra of compounds NOF1 and NOF1-Cys are
shown in Figure S1. As expected, the NOF1 probe showed a weak fluorescence due to
fluorophore quenching by the carbon–carbon double bond [34]. However, NOF1-Cys
showed significant NIR fluorescence with a maximum absorption at 670 nm and an
emission peak at 741 nm.
UV-vis absorption and fluorescence spectral changes of NOF1 (10 µM) reacting
with different Cys concentrations (from 0 to 1.4 eq.) in PBS (5 mM, pH =7.4) with 50%
DMSO (v/v) at 37 °C for 10 min (Figure S3) were investigated. The 10 µM NOF1
probe solution showed a major absorption peak at approximately 550 nm (Figure 1a).
Upon Cys addition, the intensity of the 550 nm absorption band decreased along with
an increase in the intensity of a new band at approximately 670 nm, leading to a 120
nm red shift. The growth in absorption intensity at 670 nm was attributed to
deacylation via the response of the NOF1 probe to Cys. Simultaneously, the solution
color changed from purple to blue. Thus, this phenomenon suggests that it is feasible
for Cys to be detected by the “naked-eye”. Furthermore, the absorption intensities of
the NOF1 probe with different Cys concentrations showed a linear correlation in the
range of 0–0.8 equivalents of Cys (Figure S2).
Fluorescence titration experiments of the NOF1 probe with Cys showed a gradual
increase in fluorescence intensity upon Cys addition (from 0 to 1.4 eq.) to the NOF1
solution (Figure 1b), with maximum value reaching at 741 nm under excitation at 670

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nm (Figure S3). The intensity of the emission peak at 741 nm was gradually enhanced
by the removal of the masking group through the addition-cyclization conjugation to
the acrylate moiety (Scheme 1). The plot of fluorescence intensities at 741 nm against
2
Cys concentration in the range 2–10 µM (0.2−1.0 eq.) showed a linear fit (R = 0.992)
(Figure S4), with a Cys LOD as low as 0.21 µM. The LOD was calculated using the
formula LOD = 3r/S, where r is the standard deviation of blank measurements and S
is the slope of the calibration curve. These results indicate the possibility of
quantitative Cys detection within a good linear range. Furthermore, the excitation and
emission of the NOF1 probe are both within the NIR range, thus allowing adequate
sensitivity for the detection of Cys in biological systems.
3
.3 Selectivity of NOF1 for Cys
A successful biological probe must have a suitably high selectivity. Therefore, we
first confirmed the specific selectivity of the NOF1 probe to Cys as well as to other
thiols with similar structures (Hcy and GSH). The time-dependent fluorescence
response spectra were then determined by monitoring the fluorescence intensity
changes in the reaction mixture. At 670 nm excitation, the NOF1 probe was almost
non-emissive and stable for 0–10 min (Figure 2a). In contrast, following the addition
of 2.0 eq. of Cys, the emission intensity at 741 nm showed an initial rapid increase,
reaching a maximum within 10 min, suggesting a complete sensing response. Similar
experiments using Hcy and GSH exhibited a minor increase in fluorescence, and
required a longer time to reach the maximum intensity. Furthermore, the color change
in the NOF1 solution in the presence of Cys, Hcy, and GSH could be optically
observed (Figure 2a, inset). Moreover, changes in emission spectra were also
examined, showing a similar trend (Figure 2b). Thus, the NOF1 probe can be used to
effectively distinguish Cys from Hcy and GSH.
The observed differences between Cys and both Hcy and GSH can be attributed to
the kinetic rate of the intramolecular adduct-cyclization reactions. The intramolecular
cyclization reaction with Cys leads to the formation of a seven-membered ring [4,47,
4
8], whereas that with Hcy leads to the formation of an eight-membered ring;

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therefore, the former is kinetically more favorable. In contrast, the intramolecular
cyclization reaction with GSH is sterically hindered by the bulkiness of its tripeptide,
leading to the formation of a conjugated thio-ether.
To further assess the selectivity of NOF1 toward Cys, the sensory responses to the
amino acids alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp),
glutamic acid (Glu), glutamine (Gln), histidine (His), isoleucine (Ile), leucine (Leu),
lysine (Lys), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), and
tyrosine (Tyr) were also investigated. The absorption spectra changes of NOF1 after
incubation with 10.0 eq. of the corresponding amino acid and Cys for 10 min at 37 °C
showed that only Cys exhibited an obvious red-shift of maximum absorbance from
5
52 to 670 nm (Figure 3a), corresponding to the color change from purple to blue
(Figure 3b). In addition, fluorescence spectra showed that only Cys exhibited a
marked off-on response with enhanced fluorescence at 741 nm when excited by 670
nm light; the remaining amino acids revealed only a slight fluorescence enhancement
(Figure S5). Thus, the NOF1 probe has a good selectivity toward Cys and could be
used for its specific detection.
The above results showed that the NOF1 probe undergoes NIR emission, enhanced
fluorescence, and is highly sensitive and selective toward Cys. Therefore, its
application in bio-samples was examined. Therefore, its application in bio-samples
was examined.
3
.4 Reaction mechanism
To verify the sensing mechanism proposed in Scheme S2, the UV-Vis and
fluorescence spectra of NOF1 (10 µM), NOF1+Cys (10 µM NOF1 reacting with 2.0
eq. Cys), and NOF1-Cys (10 µM) were obtained. The final UV-Vis and fluorescence
spectra were the same for both the mixture of NOF1 reacting with Cys (2.0 eq.) and
for NOF1-Cys, demonstrating that the reaction product from NOF1+Cys was
NOF1-Cys. Thus, the transformation process from NOF1 to NOF1-Cys was
confirmed by the spectra change (Figure 4). HR-MS analysis of NOF1 and the
product of NOF1+Cys further confirmed the above mechanism (Figure 5). NOF1

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+
showed a characteristic peak of m/z 534.2269 ([M+H] calculated value 534.2275).
Additionally, the NOF1+Cys product exhibited a characteristic peak of m/z 480.2185
+
(
[M+H] calculated value m/z 480.2169). Based on these results and previous reports,
we confirm that the reaction mechanism between NOF1 and Cys follows that
indicated in Scheme 2. Namely, the NOF1 probe reacts with Cys through a
conjugation addition and is followed by intramolecular cyclization to produce
NOF1-Cys accompanied by a release of fluorescence. To better understand the
photophysical properties of the NOF1and NOF1-Cys, theoretical calculations were
also performed through the time-dependent density functional theory (TD-DFT)
(Figure S6). The energies for the frontier MOs as well as their corresponding
HOMO-LUMO gaps are listed in Table S1. For NOF1-Cys, the π electrons on the
HOMO were essentially distributed in the xanthene backbone and Ar-OH group. In
contrast, the π electrons on the HOMO were mostly distributed in the xanthene
backbone and little on the Ar-O group of NOF1. The π electrons on the LUMO
primarily resided on the xanthene backbone both for NOF1 and NOF1-Cys. Herein,
the excitation from the LUMO to HUMO transition thus induced the strong
fluorescence observed for NOF1-Cys. The above results therefore successfully
demonstrate that the sensing mechanism of the NOF1 probe is responsive to Cys and
formed the NOF1-Cys.
3
.5 Application of NOF1 in the detection of Cys in human plasma
Despite the existence of fluorescent probes able to detect Cys, these have rarely
been applied in the determination of Cys in human plasma. Herein, the NOF1 probe
has been used to assess Cys in human plasma samples appropriately diluted to fit the
linear detection requirements. The average concentration of Cys in human plasma was
shown to be approximately 166.4 µM (Table 1), which is in agreement with the
reported results (130 to 290 µM Cys in human plasmas)[49,50]. Furthermore, Cys
was added to the plasma in different concentrations to determine the recovery using
the proposed method. The Cys recoveries ranged from 98% to 102% (Table 1), with a

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relative error of no more than 5%, suggesting that the proposed method has great
potential for the determination of Cys in real samples.
3
.6 Cell cytotoxic assays and cell imaging
In order to pursue its application in vivo, its cytotoxicity was assessed. HeLa cells
were treated with different concentrations of NOF1 (up to 30 µM) and the toxicity
was assessed (Figure S7). At a 10 µM NOF1 concentration, cell viability remained
unchanged after 24 h incubation, with more than 95% of cells remaining viable. When
the NOF1 concentration was increased to 30 µM, cell viability remained above 80%.
Furthermore, no sub-cellular apoptotic changes or significant cell death were
observed following incubation with working concentrations required for imaging (5
µM for cell imaging and 10 µM for in vivo imaging), with cell viability remaining
above 90%. In general, the NOF1 probe exhibited a low cytotoxicity when used
within the concentrations and incubation periods required for imaging.
In order to prove that the NOF1 probe has a practical utility for intracellular Cys
detection in living cells, confocal luminescence imaging was performed. When HeLa
cells were incubated with NOF1 (5 µM) for 30 min, an obvious intracellular NIR
fluorescence was observed (Figure 6a). However, when HeLa cells were pretreated
with N-ethylmaleimide (a known thiol trapping reagent) for 120 min, followed by
incubation with NOF1 (5 µM) for 30 min, the cells showed a weaker fluorescence
(Figure 6b). In addition, following HeLa cells pretreatment with Cys (100 µM) for
1
20 min, followed by incubation with NOF1 (5 µM) for 30 min at 37 °C, a marked
enhancement of NIR fluorescence was observed (Figure 6c). These results indicate
that the probe NOF1 possesses good membrane permeability and is able to detect
changes in intracellular Cys in living cells, holding great potential for biological
applications.
3
.7 Visualizing Cys in living zebrafish
We further investigated the ability of the NOF1 probe to visualize Cys in living
zebrafish. The 4-day-old zebrafish incubated with 5 µM NOF1 for 30 min displayed

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significant fluorescence (Figure 7a). Interestingly, the fluorescence in the zebrafish
was not uniformly distributed, with the zebrafish yolk sac showing much stronger
fluorescence. It is likely that the NOF1 probe was uniformly distributed in zebrafish,
whereas Cys was not[51]; nevertheless, current evidence does not allow us to
completely exclude the non-uniform distribution of the probe in zebrafish. Conversely,
4
-day-old zebrafish pre-incubated with 200 µM N-ethylmaleimide showed minimal
fluorescence (Figure 7b), demonstrating that the NOF1 probe is tissue-permeable and
that the fluorescence generation results from the NOF1 probe interacting with Cys in
the living zebrafish.
3
.8 Fluorescence imaging in vivo
On the basis of the probe’s NIR optical property, the NOF1 probe was explored as
a tool for imaging of Cys in the living mouse. In vivo imaging of Cys using NOF1
was performed in living BALB/c nude mice. Firstly, the living mice were
subcutaneous injected with 50 µL of 25 µM NOF1. As shown in Figure 8a, weak
emission signals of mice were detected 5 min after the injection of NOF1, and then
the fluorescence was enhanced after 30 min (Figure 8b). This was induced by the
interaction between NOF1 probe and endogenous Cys. Therefore, the above results
demonstrate that the NOF1 probe possesses the ability to sense Cys in living animals.
Conclusion
In conclusion, a novel NIR xanthene fluorescent NOF1 probe for the highly
selective and sensitive detection of Cys over Hcy, GSH, and other amino acids was
synthesized. The addition-cyclization conjugation reaction of Cys toward the acrylate
moiety in the NOF1 probe results in the cleavage of the acrylate moiety and release of
a free phenolic OH, thereby inducing a significant enhancement of fluorescence. This
was confirmed by spectral changes and HR-MS. Importantly, the NOF1 probe
exhibited a high selectivity and sensitivity towards Cys with a low LOD of 210 nM in
vitro, as well as allowing the imaging of Cys in living cells, zebrafish, and in vivo.
Therefore, this novel probe has great potential for applications involving the detection

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of Cys both in vitro and in vivo. The present results offer valuable information and
create a new platform for the design of NIR-emitting probes based on xanthene
derivatives for fluorescence detection and imaging in vivo.
Acknowledgments
This work was supported by Natural Science Foundation of China (No. 21501031
and 51478123), and Hong Kong Polytechnic University (1-ZE1C and 1-YW2T). Wu
et al. gratefully acknowledges Dr. Huiqiang Lu of Jinggangshan University for
experimental support in zebrafish imaging.
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List of Captions
Scheme 1. Chemical structure of NOF1, and the process of detection of Cys in vitro
and in vivo
Scheme 2. Proposed mechanism for sensing of NOF1 for Cys
Figure 1. (a) UV-vis absorption and (b) fluorescence spectral changes of NOF1 (10
µM) upon addition of different concentrations Cys (from 0 to1.4 eq.) in PBS(5 mM,
pH=7.4) with 50 % DMSO (v/v) at 37 °C for 10 min. Inset: Color changes in NOF1
upon addition of Cys (10 µM), λ =670 nm
ex
Figure 2. (a) Time course of the fluorescence response at 741 nm. Inset: color
changes in NOF1 upon addition of Cys, Hcy, and GSH (2.0 eq.). (b) Emission
spectral changes of NOF1 in the absence (blank) and presence of Cys, Hcy, and GSH
(
2.0 eq.) in PBS (5 mM, pH=7.4) with 50% DMSO (v/v). [NOF1] = 10 µM, λ = 670
ex
nm, 37 °C
Figure 3. (a) Absorption spectra of probe NOF1 (10 µM) treated with various
analytes (10.0 eq.). (b) Color change of probe NOF1 (10 µM) treated with 10.0 eq. of
various analytes. All experiments were performed in PBS (5 mM, pH=7.4) with 50%
DMSO (v/v) at 37 °C for 10 min
Figure 4. (a) Absorption and (b) fluorescence spectra of NOF1 (10 µM) and the
product of NOF1 (10 µM) reacted with Cys (1.0 eq.) and NOF1-Cys (10 µM) in PBS
(
5 mM, pH=7.4) with 50 % DMSO (v/v), λ = 670 nm
ex
Figure 5. HR-MS spectrum of NOF1 (left side) and NOF1+Cys (right side)
Figure 6. Confocal luminescence images of living HeLa cells. (a) HeLa cells
o
incubated with NOF1 (5 µM) for 30 min at 37 C. (b) HeLa cells were pretreated with
NEM (100 µM) for 120 min and then further incubated with NOF1 (5 µM) for 30 min
at 37 °C. (C) HeLa cells were pretreated with Cys (100 µM) for 120 min and then
o
further incubated with NOF1 (5 µM) for 30 min at 37 C. Emission was collected by
an NIR channel at 655−755 nm, under excitation with 635 nm laser. Scale bar = 30
µm

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Figure 7. In vivo images of zebrafish treated with NOF1. (a) 4-day-old zebrafish was
incubated with 10 µM NOF1 for 30 min. (b) 4-day-old zebrafish was pre-incubated
with 200 µM NEM for 30 min and then incubated with 10 µM NOF1 for 30 min
Figure 8. Fluorescence imaging of endogenous Cys in living mice injected with
NOF1 (25 µM, 50 µL) in saline at 5 min (a) and 30 min (b) after subcutaneous
injection. Emission was collected by an NIR channel at 660−780 nm, under excitation
with 635 nm laser.
Table 1. Determination of Cys in human plasmas with probe NOF1

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Table 1 Determination of Cys in human plasma with probe NOF1
a
a
Analyte in plasmas
Added
found
(µM)
Recovery
(µM)
(%)
－
b
Cys
0
2
4
6
1.04 ±0.05
3.11±0.03
5.05±0.03
7.12±0.02
102±2.0
98±1.1
101±3.0
a
Mean of three determinations ± standard deviation
b
Mean of three determinations after human plasma diluted 160-fold

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Scheme 1. Chemical structure of NOF1, and the process of detection of Cys in vitro
and in vivo

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Scheme 2. Proposed mechanism for sensing of NOF1 for Cys

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Figure 1. (a) UV-vis absorption and (b) fluorescence spectral changes of NOF1 (10
µM) upon addition of different concentrations Cys (from 0 to1.4 eq.) in PBS(5 mM,
pH=7.4) with 50 % DMSO (v/v) at 37 °C for 10 min. Inset: Color changes in NOF1
upon addition of Cys (10 µM), λ =670 nm
ex

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Figure 2. (a) Time course of the fluorescence response at 741 nm. Inset: color
changes in NOF1 upon addition of Cys, Hcy, and GSH (2.0 eq.). (b) Emission
spectral changes of NOF1 in the absence (blank) and presence of Cys, Hcy, and GSH
(
2.0 eq.) in PBS (5 mM, pH=7.4) with 50% DMSO (v/v). [NOF1] = 10 µM, λ = 670
ex
nm, 37 °C

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Figure 3. (a) Absorption spectra of probe NOF1 (10 µM) treated with various
analytes (10.0 eq.). (b) Color change of probe NOF1 (10 µM) treated with 10.0 eq. of
various analytes. All experiments were performed in PBS (5 mM, pH=7.4) with 50%
DMSO (v/v) at 37 °C for 10 min