A near-infrared fluorescent probe for the discrimination of cysteine in pure aqueous solution and imaging of cysteine in hepatocellular carcinoma cells with facile cell-compatible ability

Article abstract of DOI:10.1039/c9nj00129h

A unique near-infrared fluorescence sensor named AySA was designed and synthesized for selectively detecting cysteine over Hcy and GSH in aqueous solution with fast response and long emission wavelength of 701 nm. In this strategy of designing the probe, sulfonic acid was introduced to the cyanine structure through multiple methylene flexible chains as the water soluble functional group, and an acryl group was introduced through nucleophilic substitution reaction to serve as an efficient reaction site for Cys. Finally, a new NIR fluorescence sensor was constructed for the discrimination of Cys in aqueous solution. The probe shows excellent properties in 100% aqueous solution. It shows evident discrimination of Cys over Hcy and GSH, which is accompanied with fluorescence intensity enhancement and visible color change. The detection limit was calculated for 21.1 nm using the linear relationship of concentration of 1-3 μM (3σ/κ). The proposed mechanism based on conjugated addition, intramolecular cyclization and the sensing mechanism was confirmed by high-resolution mass spectrometry. In addition, the probe can work at a wide range of pH 7-10 and react with Cys in a few minutes. The probe was also successfully applied to Bel 7402 imaging with low toxicity. An outstanding red fluorescence appeared when the Bel 7402 cells were incubated with the probe or when Cys was added to the NEM-pretreated cells, indicating that the probe has a good ability to penetrate cells and react with Cys quickly. Considering the above results, we have the confidence to say that the probe can be used as an ultra-sensitive, near-infrared fluorescence sensor to selectively sense Cys in pure aqueous solutions in living systems.

Full text of DOI:10.1039/c9nj00129h

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ISSN 1144-0546
PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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A near-infrared fluorescent probe for the discrimination of
cysteine in pure aqueous solution and imaging of cysteine in
hepatocellular carcinoma cells with facile cell-compatible ability
Received 00th January 20xx,
Accepted 00th January 20xx
Xin Yang, Ying Qian*
A unique near-infrared fluorescence sensor named AySA was designed and synthesized for selectively detecting cysteine
over Hcy and GSH in aqueous solution with fast respond and long emission wavelength to 701 nm. In this strategy of
designing the probe, a sulfonic acid was introduced to the cyanine structure through multiple methylene flexible chains as
the water soluble functional group and acryl group was introduced through nucleophilic substitution reaction for using as
an efficient reaction site for Cys. Finally, a new NIR fluorescence sensor for discrimination of Cys in aqueous solution was
constructed. The probe shows excellent properties in 100 % aqueous solution. It shows obvious discrimination of Cys over
Hcy and GSH accompanied with fluorescence intensity enhancement and visible color change. The detection limit was
calculated for 21.1 nm using the linear relationship of concentration of 1-3 μM (3 σ/κ). The proposed mechanism based on
conjugated addition and intramolecular cyclization and the sensing mechanism was confirmed by high resolution mass
spectrometry. In addition, the probe can work at a wide range of pH 7-10 and react with Cys in a few minutes. The probe
was also successfully applied to Bel 7402 imaging with low toxicity. There was an outstanding red fluorescence occurred
whether the Bel 7402 cells was incubated with probe or the NEM-pretreated cells added Cys, the result reveals that the
probe has good ability to penetrate cells and reacted with Cys quickly. Considering of above result, we have the confidence
to say the probe can be used as an ultra-sensitive, near-infrared fluorescence sensor for selectively sense Cys under pure
DOI: 10.1039/x0xx00000x
www.rsc.org/
aqueous
solutions
in
living
system.
poor solubility of fluorophor results in probes with poor sensitivity
and difficulty in finding real-life applications. Moreover, there were
few reports on probes are capable of specifically distinguishing Cys
from other amino acids in a pure water system. Using water as a
solvent can avoid the influence of this factor, it is extremely
important to develop a fluorescent sensor that recognizes Cys in
pure aqueous solution. J.-J. Zhang22 had developed a fluorescence
sensor based on cyanine dye for selectively sense Cys in H2O/DMSO
solution. J.-C. Xu et al23 developed a new fluorescence probe for
detecting Cys and GSH from different channels and applied in cell
imaging. And the majority of those probes can’t distinguish
Introduction
Cysteine is an important amino acid containing a free thiol
moiety and is of great value for many cellular functions in the living
system.1-6. The development of fluorescence sensor for cysteine has
attracted great attention as cysteine plays a vital role in in biological
processes contribute to many physiological processes, such as the
intracellular redox activity, xenobiotic metabolism, and intracellular
signal transduction7-12. The evidence shows that the deficiency of
cysteine is related to some disease including delayed growth, liver
damage, fat loss and even skin lesions13-19. As a result, fluorescence
sensors owing to their sensitivity and simplicity have attracted
considerable attention in recent years20-21. Nevertheless, most of
the probes for cysteine detection were used in organic solvent
rather than pure aqueous solution. It is necessary for the researcher
to develop a water-soluble fluorescence transducer detecting
cysteine in pure aqueous solution rather than organic solvents. It
makes this probe safer and more widely used in living systems.
The choice of fluorophores with sufficient brightness and
photostability is critical to the development of such probes. The
Hcy/Cys/GSH from each other owing to their structural similarity.24-
29
Compared with another paper, J.-O. –Yang30 developed
a
rhodamine fluorescence probe with high water solubility for detect
GSH and applied in cell imaging. However, the absorption and
emission wavelength was seated in visible light range (300–650 nm),
which hinder the application of near infrared imaging. 31-35. Recently,
NIR fluorescent probes were developed to take advantage of near-
infrared imaging unique features such as deep tissue penetration,
small back ground interference and small damage to the sample
and low biological luminescence and so on36-44. There were several
characteristics for develop a high performance optical molecular
probe including high brightness, water solubility, low cytotoxicity
and photo stability. Thus, the development of a new near-infrared
fluorescent probe which could selectively detect Cys / Hcy / GSH in
pure aqueous solutions is a challenge for researchers.
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In this paper, a new near-infrared fluorescence probe was TOF) spectrometer. UV–visible absorption spectra and fluorescence
View Article Online
devised for identify Cys in pure aqueous solution, using sulfonic acid spectra were accessed by Shimadu UV-36D0O0I:s1p0e.1c0t3ro9/pCh9oNtJo0m0e12t9eHr.
as a water-soluble functional group ,hemi-cyanine dye as
a
and HORIBA FL-4 Max spectrometer.
fluorophore and acrylate group as a recognition receptor to detect
All the reagents were purchased from Macklin Biochemical
cysteine based on conjugated addition. Compound of probe AySA is Co.,Lttd (Shanghai, China), Aladdin ,Maya reagent company or
composed of a sulfonic acid as a water-soluble functional group and Sigma-Aldrich and used as received. The solvents used in the
an acrylate group as the quenching and recognizing moiety, caged synthesis and spectroscopy tests are of analytical grade.
NIR hemi-cyanine dye with a zwitterionic structure. The results
Mother liquor of probe AySA in pure PBS was prepared before
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demonstrated that the probe showed highly discrimination for Cys used. The amino acids solutions, including Cys, Hcy, GSH, Phe, Ala,
from Hcy and GSH within a few minutes in 100% aqueous solutions Pro, Gly, Glu, Gln, Arg, Lys, Tyr, Trp, Asp, Asn, His were also
and long emission wavelength to 701 nm. For one thing, it has been prepared with the concentration of 1 ×10-2 M.
applied to the imaging of Bel 7402 cells imaging, the clear red General measurement parameters
imaging illustrated that it has good ability penetrating into cells and
The stock solution of probe various amino acids were prepared
reacted with intracellular cysteine in a few minutes. In summary, by dissolving them in pure PBS to a concentration of 0.1 mM and 10
the designed probe has great application prospects for the mM. The test solutions were prepared adding appropriate aliquots
detection of biothiols in tissues and animals owing to the excellent of each amino acid to the diluted AySA solution (10 mM).
properties showed in pure aqueous solution.
The absorption and fluorescence spectra titrations of AySA
dosimeter were operating in pure PBS solution and the detection
limit is then calculated using the following equation. Detection limit
= 3 σ/κ, Where σ is the standard deviation of blank measurement
and κ is the slope between the emission intensity and concentration.
The quantum yield (Φs) was calculated using the following
equation45-47 ΦF(X) = ΦF(S) (ASFX/AXFS) (nX/nS)2. Where A is the
absorbance at the excitation wavelength, F is the area under the
corrected emission curve, and n is the refractive index of the
solvents used. Subscripts X and S refer to the unknown and the
standard, respectively. Fluorescence quantum yield was determined
by indocyanine green (ICG, Φf = 0.13) in DMSO using the literature
method as a reference48
Recent literatures for Cys/ Hcy/GSH in mixed solvent solution
MTT cytotoxicity assay
An in vitro MTT assay was used to assess the toxicity of the probe
AySA. First, Bel-7402 cells were seeded in a 96-well plate and
cultured at 37 °C, 5 % CO2 for 24 hours to allow cells to adhere.
Then different concentrations of the probe AySA solution were then
added to the above cells, and incubation was continued for 24
hours under the same conditions. Subsequently, the cells were
treated with 5 mg / mL MTT and cultured for 4 hours. After the
above suspension was finally discarded, 100 μl of DMSO was added
to each well, and the mixture was shaken uniformly for direct test
on a Benchmark Plus plate reader to record the absorbance of each
hole in a 96 well plate at 570 nm. The toxicity of the probe molecule
AySA was assessed by cell viability. The formula is as follows: Cell
viability(%)=Experimental value OD570/ Control value OD570
×100%, where OD570 is the absorbance measured at 570 nm.
This probe work in pure aqueous solution:
Pure water solution
Pure water solution
Scheme 1: Recent reports for detecting GSH, Hcy and Cys in mixted solution,
structure of probe AySA and specific recognition of Cys; the left is the
structure of probe and the right is the recognized product, after Cys was
added, the strong red fluorescence occurred.
Cell culture and NIR imaging
The Bel-7402 cells were seeded in a glass bottom copolymer
culture dish and cultured at 37 ° C, 5% CO 2 for 24 hours in order to
keep the cells from being suspended. The medium was 10% fetal
bovine serum and 90% 1640 medium. Subsequently, a specific
concentration of the probe solution prepared in DMSO was added
to the above glass confocal culture dish, and it was incubated with
the cells for 30 minutes. At last, the cells were washed three times
with PBS and directly subjected to fluorescence imaging on a
confocal laser scanning microscope. (FLUOVIEW FV3000. OLYMPUS).
Experimental part
General methods and reagent
1H NMR and 13C NMR spectra were recorded on Bruker DMX300
NMR spectrometer and Bruker AV II–400 MHz spectrometer in
DMSO-d6, Tetramethylsilane (TMS) was used as the internal
standard. Mass spectra were recorded on the Ultraflex II (MALDI-
2 | J. Name., 2012, 00, 1-3
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dihydro-1H-xanthen-4-yl)vinyl)-3,3-dimethyl-1-(4-sulfobutyl)-3H-
Synthesis
The synthetic route of AySA was illustrated in Scheme 2. The
probe molecules AySA were synthesized in five steps. Among them,
intermediate M1 and intermediate M2 are synthesized according to
the synthetic steps reported in the literature.49-50
Synthesis of M1 Add phenylhydrazine (9.82 mL, 0.10 mol)
and 3-methylbutan-2-one (12 mL, 0.11 mol,) to a 50 ml round
bottom flask, use ethanol (30 mL) as a solvent, 3 drops of acetic
acid as a catalyst, and stir the reaction at 50 °C for 4 hours. After
completion of the reaction, the solvent was removed in vacuo and
then acetic acid (30 mL) was added, reacted at 80 °C for 1 hour. The
acetic acid was distilled off under reduced pressure, and the residue
was diluted with methylene chloride, dried over anhydrous sodium
sulfate and then purified by column chromatography using Ethyl
acetate: petroleum ether (1:10) to afford the yellow liquid 2,3,3-
trimethyl-3H-indole 15.1 g. Yield: 95 %.
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1
DOI: 10.1039/C9NJ00129H
indol-1-ium as the Blue- Green solid. (0.38 g, Yield: 75 %). H NMR
(300 MHz, DMSO-d6): δ 8.44 (d, J = 7.2 Hz, 1 H), 7.96(s, 1 H), 7.69 (d,
J = 3.6 Hz, 1 H), 7.61(d, J = 4.2 Hz, 1 H), 7.55(s, 1 H), 7.49~7.44(m, 2
H), 7.37(d, J1 = 7.5 Hz, J2 = 3.9 Hz, 1 H), 6.82 (s, 1 H), 7,79~6.77(m, 1
H), 6.41 (d, J = 7.5 Hz, 1 H), 6.19~6.17(m, 1 H), 4.32(t, J1 = 3.6 Hz, J2 =
3.6 Hz, 2 H), 2.89(s, 2 H), 2.73(s, 2 H), 2.70~2.66(m, 4 H), 2.57(t, J1 =
3.6 Hz, J2 = 3.0 Hz, 3 H), 1.91 (s, 1 H), 1.70 (s, 6 H)
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The synthesis of AySA: Intermediate M4(0.25 g, 0.5 mmol) and
acryloyl chloride(8 μL,1 mmol) were added to a 50 mL round
bottom flask containing 10 mL of anhydrous dichloromethane
under ice bath for 20 min. After the reaction is completed, the
dichloromethane and unreacted acryloyl chloride are removed by
rotary evaporation, and the crude product is purified by column
chromatography using Petroleum ether: dichloromethane: ethanol
=10:10:1 as the eluent to obtained product (E)-2-(2-(6-(acryloyloxy)-
2,3-dihydro-1H-xanthen-4-yl)vinyl)-3,3-dimethyl-1-(4-sulfobutyl)-
3H-indol-1-ium as purple solid (0.13 g, 46 %) 1H NMR (300 MHz,
DMSO-d6): δ 8.48 (d, J = 7.5 Hz, 1 H), 7,72 (d, J = 3.9 Hz, 1 H), 7.64 (d,
J = 3.9 Hz, 1 H), 7.54(s, 1 H), 7.50(t, J1 = 3.9 Hz, J2 = 3.9 Hz, 1 H),
7.46( d, J = 1.8 Hz, 1 H), 7.39 (t, J1 = 3.6 Hz, J2 = 3.9 Hz, 1 H)，6.87(s,
1 H), 6.82~6.81(m, 1 H), 6.45 (d, J = 7.5 Hz, 1H), 4.35(t, J1 = 3.6 Hz, J2
= 3.6 Hz, 2 H), 2.71~2.66(m, 4 H), 2.57~2.54(m, 2 H), 2.02~1.97(m, 1
H), 1.71(s, 6 H), 1.34(s, 1 H), 1.23(s, 8 H) 13C NMR (DMSO-d6):179.61,
165.07, 164.09, 157.22, 148.57, 147.50, 144.73, 137.45, 133.52,
132,22, 129.48, 128.79, 125.75, 117.83, 117.40, 117.02, 116.07,
106.43, 104.99, 53.75, 43.49, 31.38, 30.37, 29.36, 26.65, 25.56,
23.11, 11.52. HRMS: calue for 560.21013, Found : 560.21063
The synthesis of M2: To a 50 ml round bottom flask was added
2,3,3-trimethyl-3H-indole (2.0 g, 12.56 mmol) and 1,2-oxathiane
2,2-dioxide (5.47 g 40.19 mmol) using dry dichlorobenzene as the
◦
solvent and reacted at 120 C for 12 h under N2 inert atmosphere.
When the reaction was completed, the mixture was poured into
100 mL ether.
A pink solid 4-(3,3-dimethyl-3H-indol-1-ium-1-
yl)butane-1-sulfonate was then obtained and used without further
purification.
The synthesis of M3
:
4-(3,3-dimethyl-3H-indol-1-ium-1-
yl)butane-1-sulfonate (5.0 g, 16.94 mmol) and (E)-2-chloro-3-
(hydroxymethylene)cyclohex-1-enecarbaldehyde (1.45 g, 8.47
mmol) and anhydrous sodium acetate (0.69 g, 8.47 mmol) were
added to the 50 mL round bottom flask using acetic anhydride (20.0
mL) as the solvent and refluxed at 90 ◦C for 1 h under N2
atmosphere. When the reaction was completed, it was cooled to
room temperature, the solvent was removed under reduced
pressure, and then purified by column chromatography using
CH2Cl2/ 2% Ethanol to get product 2-((E)-2-((E)-2-chloro-3-((E)-2-
(3,3-dimethyl-1-(4-sulfobutyl)indolin-2-
ylidene)ethylidene)cyclohex-1-en-1 yl)vinyl)-3,3-dimethyl-1-(4-
sulfobutyl)-3H-indol-1-ium as a blue solid 3.07 g Yield: 50%. 1H
NMR (300 MHz, DMSO-d6): δ 8.27 (d, J = 6.9 Hz, 2 H), 7.63 (d, J = 3.9
Hz, 2 H), 7.50 (d, J=3.9 Hz, 2 H), 7.44~7.41 (m, 2 H), 7.29~7.27 (m, 2
H), 6.39 (d, J=6.9 Hz, 2 H), 4.38 (t, J1 = 2.4 Hz, J2 = 4, 1 H), 4.24 (t, J1 =
3.6 Hz, J2 = 3.9 Hz, 4 H), 3.45 (m, 2 H), 2.74 (t, J1 = 3.0 Hz, J2 = 3.0 Hz,
4 H), 1.86~1.74 (m, 10 H), 1.67 (s, 12 H), 1.07 (t, J1 = 3.3 Hz, J2=3.6
Hz, 3 H)
The synthesis of M4: To a 50 mL round bottom flask was added
resorcinol (0.22 g, 2.0 mmol) and K2CO3 using acetonitrile (15 mL) as
the solvent and stirred at room temperature for 30 mins under N2
atmosphere. A solution of compound M3(0.73 g, 0.1 mmol) in N,N-
dimethylformamide (10 mL) was added to the above mixture using
constant pressure dropping funnel. Then, the mixture was heated at
50 ° C for 4 hours. When the reaction was completed, acetonitrile
and N.N-dimethylformamide were removed under reduced
pressure, and the crude product was purified by column
chromatography using Petroleum ether: dichloromethane: ethanol
=10:10:1 as the eluent to get the product (E)-2-(2-(6-hydroxy-2,3-
Results and discussion
3.1 The design concept and synthesis of Water-soluble
fluorescent probe AySA
A novel near-infrared fluorescence sensor with an emission
wavelength of 701 nm and a response speed of 2 min was designed
by linking the acrylate group to the hemi-cyanine skeleton for
selectively detection Cys over Hcy and GSH under pure water
conditions. The water-soluble hemicyanine probe AySA was taken
five steps to synthesis. A sulfonic acid was introduced to the
cyanine structure through multiple methylene flexible chains as the
water soluble functional group. It was linked to the AySA through
substitution reaction and acrylate group was introduced as a
recognition receptor to detect cysteine based on conjugated
addition through nucleophilic substitution reaction. The skeleton of
the hemi-cyanine allows the absorption and emission wavelengths
of the probe AySA reach to the near-infrared region. Eventually,
novel near-infrared fluorescence sensor with an emission
wavelength of 701 nm and fast respond of 2 min was constructed
for detecting cysteine under the condition of 100 % aqueous
solution. It was successfully applied in Bel 7402 cell imaging with
low toxicity.
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Both the intermediate and the target product passed the compared with blank solution. The experiment resulVtieiwmAprtliiceledOtnhlianet
DOI: 10.1039/C9NJ00129H
nuclear magnetic resonance, carbon and high resolution mass the probe was shown highly selectively toward Cys over Hcy and
spectrometry.( Supporting information, Fig. S1 – S5 )
GSH.
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(a)
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Scheme 2 The synthesis route of AySA
(b)
3.2 The highly selectivity of probe for cysteine in aqueous solution
The probe AySA can achieve naked eye recognition of cysteine in
aqueous solution and react with Cys in a few minutes. As the
picture (Fig.S6) vividly described that, its color changes have a
dramatic response when adding the Cys to the blank solution. The
solution of the probe molecule is blue-violet, after the cysteine was
added to the solution, the color was changed to indigo, there was
no similar change when Hcy/GSH and the other amino acids
including Phe, Ala, Pro, Gly, Glu, Gln, Arg, Lys, Tyr, Trp, Asp, Asn, His
added to the blank solutions. This phenomenon illustrated that the
probe can selectively sense cysteine in pure aqueous solution.
Then, we test the sensing behaviour of probe AySA for Cys under
the condition of pure water solution. As shown in the Fig.1(a), there
was an significant enhancement when Cys was added to the
solution with the emission wavelength located at 701 nm, however,
Fig.1 (a) The emission fluorescence intensity was normalized. The relative fluorescence
intensity shown in the figure was obtained by dividing the fluorescence intensity at 701
nm by the fluorescence intensity of the blank probe. The fluorescence response of
probe (10 μM) in the presence of various amino acids including Cys, Hcy, GSH, Phe, Ala,
Pro, Gly, Glu, Gln, Arg, Lys, Tyr, Trp, Asp, Asn, His (10 μM) in PBS buffer solution (pH =
7.4) (b) The absorbance response of probe (10 μM) in the presence of various amino
acids (10 μM) in PBS buffer solution (pH = 7.4)
a
blank solution of probe shows no obvious enhancement
compared with when the Hcy and GSH Phe, Ala, Pro, Gly, Glu, Gln,
Arg, Lys, Tyr, Trp, Asp, Asn, His added to the solution. Selective
experiments have shown that the probe has significant selectivity
for Cys and can be used for complex biological sample testing. The
UV spectrum was also conducted as the Fig.1(b) illustrated that, the
absorption band of blank solution located at 592nm、619 nm and
669 nm. A significant red shift in absorption wavelength was
observed when Cys was added to the blank solution and the
absorption band was shift to 603 nm 652 nm and 678 nm. Molar
extinction coefficient was calculated to 1.14 × 104 M-1 cm-1 at 652
nm accompanied the color change from blue-violet to cyan and the
maximum absorption peak of the ultraviolet absorption spectrum
will greatly shift after the action of probe and Cys. While there was
a small or no change when GHS, Hcy and Phe, Ala, Pro, Gly, Glu, Gln,
Arg, Lys, Tyr, Trp, Asp, Asn, His were added to the solution
Moreover, in order to investigate whether the probe can still
have good selectivity and anti-interference ability to Cys in complex
systems, interference test was carried out in pure aqueous solution.
We did the competitive experiments to show that the probe can
still recognize Cys in the presence of other amino acids. As the
Fig.S7 shows that in presence of the above –mentioned interfering
substance, the probe did not respond to them, while, the Cys is still
well detected. Obviously, all these results indicated that AySA has
good selectivity low concentration of Cys rather than high
concentration of glutathione and homocysteine in pure aqueous
solution.
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3.3 Sensing modes based on the conjugated addition and unsaturated carbonyl group, the intermediate thioether was then
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DOI: 10.1039/C9NJ00129H
generated. Subsequently, the intramolecular cyclization reaction
intramolecular cyclization
Considering the above results, a possible response mechanism giving
desired
lactam
and
compounds
M4.
of probe discriminating Cys from Hcy and GSH under the condition In order to further confirm the sensing mechanism, the high
of organic solvent free recognize system was proposed as shown in resolution mass spectrometry was carried out. As the Fig.3
Fig.2. Acrylate group was introduced to the hemicyanine compound described that, When 10 μM Cys was added to the blank solutions,
as the reaction sites for Cys through nucleophilic substitution there was a new peak appeared at 528.18196, this can be
reaction. The proposed mechanism of probe identified Cys based on contributed to compound M4+Na=528.18151, which could identify
the conjugated addition of sulfhydryl group in Cys attacked the α,β- the mechanism of probe toward cysteine.
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Fig.2. Proposed mechanism for the response of probe AySA to Cys based on conjugate addition and intramolecular cyclization
equation of ΦF(X) = ΦF(S) (ASFX/AXFS) (nX/nS)2, it was highly than the
reported literature.
[M4+Na+]=528.18151
Fig.4. Fluorescence titration spectra of probe (10 μM) in pure water solution (PBS, pH =
7.4) after the concentration of Cys (0-10 μM) was added (λex = 670 nm, λem = 701 nm).
Fig.3 HRMS spectrum of probe AySA toward Cys
3.4 Sensing behaviour of probe toward Cys in pure aqueous
solution
Moreover, time and pH-dependence in the detection process of
cysteine was also carried out. The effect of reaction time on the
fluorescence intensity of the probe was performed in 100%
aqueous solution without organic solvents media assistance. As the
Next, we tested the Cys response ability of the probe under the
condition of organic solvent free recognize system. When adding
different concentrations to the probe test system, the fluorescence Fig.5 shows, once 10 μM cysteine added to the solution, there was
intensity was also enhanced accompanied with the increase
concentration of Cys. The result reveals that with the
concentrations of the probe increased, the fluorescence intensity
was also gradually enhanced at 701 nm and saturated at 10 eq. At
the same time, we also observed a good linear relationship with the
concentration of 1-3 μM, the detection limit was calculated for 21.1
nm (3σ/k). The quantum yield was calculated for 7.6 % using the
an remarkable fluorescence intensity increase at 701 nm and
reached equilibrium within 24 min. This outcome can be
contributed to that the nucleophilic attack addition of the double
bond to the sulfhydryl group in the probe, the ortho-amino group
further attacks the ester carbonyl group to form a ring, wherein Cys
is a favorable seven-membered ring. This is also can interpreted
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that why the probe can recognize Cys in a short time. To
demonstrate whether probe can still detect Cys in a physiological
pH environment, we performed a probe response to Cys in different
pH buffers. As the Fig.S8 described that, it was found that the probe
was very stable between pH = 2~6 in presence or absence of
cysteine. After pH>6, the fluorescence intensity increased slightly,
this phenomenon may be caused by hydrolysis of the ester group
under alkaline conditions to release M4. There was a significant
enhancement of the fluorescence intensity under a wide range of
pH6-10 in presence or absence of Cys, which was suitable for
detecting Cys under physiological pH conditions.
View Article Online
DOI: 10.1039/C9NJ00129H
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Fig.6 Cytotoxicity of Probe in Bel 7402 cells. Cells were treated with different
concentrations of Probe for 24 h, and the cell viabilities were determined by the
MTT assay. Data are expressed as the mean ± SD
Based on the above results, the selective fluorescent imaging
of Cys in living cells was investigated. Endogenous and exogenous
fluorescence imaging of the probe was also included. As shown in
the Fig. 7(a-c), a significant red fluorescence was observed when
the Bel 7402 cells was co-incubation with probe AySA under the
◦
condition of 37 C for 30 min. The result indicates that the probe
has the ability to permeate into cells and react with endogenous
Cys to generate red fluorescence. In contrast, Fig.7(d-f) shows no
fluorescence after the cells pretreat with 0.5 mM N-ethylmaleimide
for 30 min and incubated with the probe for another 30 min. Finally,
the Cys(5 mM) was added to the NEM-pretreated Bel 7402 cells, a
Fig.5. Time-dependent fluorescence intensity of probe (10 μM) in PBS buffer solution
(pH = 7.4) with 10 μM of Cys.
3.5 Cell imaging and possible detection of intracellular Cys A marked stronger red fluorescence was observed from Fig.7(g-i). The
conventional MTT assay was performed to verify whether the probe result indicating that the probe is easy to enter the cells as a
AySA has sufficiently low toxicity to detect biomolecules in living fluorescence label for specific detection of Cys. The low toxicity of
cells by using cell viability as a standard. As shown in Fig. 6, the cells the probe may render it applicable for imaging in living systems. It
was treated with different concentration of probe and co-incubated also could be further applied in tracing and detecting abnormal
◦
under the condition of 5 % CO2, 37 C for 24 h. The result showed content of cysteine in biological system and achieve the goal of
that there was no apparent loss of cell viability under the maintaining normal human activities. It has the great potential in
experimental conditions. This reveals that the probe has low tissue and in vivo imaging.
cytotoxicity to the cultured cells under the experimental condition
and the probe has great potential in cell imaging study.
Compared with the other fluorescence probe for
determination of Cys, this probe AySA could be applied in pure
aqueous solution rather than organic solution. The majority of
previously reported probes are performed in either pure organic or
organic-aqueous solutions, which limits their practical applications
in a physiological environment. The proposed method for construct
probes selectively sense Cys under the organic free recognize
system was used for the determination of Cys in real samples and
living system.
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addition, the probe can work at a wide range of pH(7V-1ie0w)Aartnicdle Orenalincet
DOI: 10.1039/C9NJ00129H
with Cys in a few minutes. The probe was also successfully applied
to Bel 7402 imaging with low toxicity. There was an outstanding red
fluorescence occurred whether the Bel 7402 cells was incubated
with probe or the NEM-pretreated cells added Cys, the result
reveals that the probe has good ability to penetrate cells. Consider
of above result, we have the confidence to say the probe can be
used as an ultra-sensitive fluorescence sensor for selectively sense
Cys under pure aqueous solutions in living system. It is a challenge
for the researcher to use this strategy to improve the water
solubility of NIR fluorescent probe through structural modification.
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Merge
Red channel
Bright field
(a)
(b)
(e)
(c)
(f)
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(d)
(g)
Conflicts of interest
There are no conflicts to declare.
(i)
(h)
Acknowledgements
We thank the financial support from the Fundamental Research
Funds for the National Natural Science Foundation of China (No.
61178057).
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AySA was designed and synthesized for the discrimination of
cysteine over Hcy and GSH under the condition of pure aqueous
solution with fast responds for 2 min and long emission wavelength
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cyclization occurred to give two compounds. The sensing
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DOI: 10.1039/C9NJ00129H
The table of contents entry
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Manuscript ID NJ-ART-01-2019-000129
Title: A near-infrared fluorescent probe for the discrimination of cysteine in pure aqueous
solution and imaging of cysteine in hepatocellular carcinoma cells with facile cell-compatible
ability
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Pure water solution
Pure water solution
A NIR fluorescence sensor for selectively detecting cysteine in aqueous solution with fast
respond and long emission wavelength was synthesized.