An ESIPT coupled AIE fluorescent probe for biothiols detection and imaging based on a chalcone fluorophore

Article abstract of DOI:10.1016/j.dyepig.2020.108627

Biothiols are closely related to numerous physiological and pathological processes in living organisms and cells. Fluorescent probes are powerful tools for sensitive detection and imaging of biothiols in biological samples. In this work, we developed a no

Full text of DOI:10.1016/j.dyepig.2020.108627

Journal Pre-proof
An ESIPT coupled AIE fluorescent probe for biothiols detection and imaging based on
a chalcone fluorophore
Fangfang Dai, Min Zhao, Fangzhou Yang, Tingting Wang, Chao Wang
PII:
S0143-7208(20)31324-3
DOI:
https://doi.org/10.1016/j.dyepig.2020.108627
DYPI 108627
Reference:
To appear in: Dyes and Pigments
Received Date: 22 March 2020
Revised Date: 3 June 2020
Accepted Date: 3 June 2020
Please cite this article as: Dai F, Zhao M, Yang F, Wang T, Wang C, An ESIPT coupled AIE fluorescent
probe for biothiols detection and imaging based on a chalcone fluorophore, Dyes and Pigments, https://
doi.org/10.1016/j.dyepig.2020.108627.
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Author Statement
Dai Fangfang: Methodology, Investigation, Writing- Original draft. Zhao Min: Investigation,
Writing- Original draft. Yang Fangzhou: Investigation, Wang Tingting: Investigation, Wang
Chao: Conceptualization, Writing - Review & Editing, Supervision, Funding acquisition.

Graphical abstract

An ESIPT coupled AIE fluorescent probe for biothiols detection and
imaging based on a chalcone fluorophore
Fangfang Dai, Min Zhao, Fangzhou Yang, Tingting Wang, Chao Wang*
College of Chemistry and Chemical Engineering, Shaanxi Key Laboratory of Chemical
Additives for Industry, Shaanxi University of Science and Technology, Xi’an 710021, China
*Corresponding author.
E-mail address: wangchao@sust.edu.cn (C. Wang)
1

Abstract:
Biothiols are closely related to numerous physiological and pathological processes
in living organisms and cells. Fluorescent probes are powerful tools for sensitive
detection and imaging of biothiols in biological samples. In this work, we developed a
novel turn-on fluorescent probe DNBS-HCA for biothiols detection based on the
simple 2′-Hydroxychalcone (HCA) fluorophore, which possesses an excited-state
intramolecular proton transfer (ESIPT) coupled aggregation-induced emission (AIE)
characteristic. The 2′-hydroxyl group of HCA fluorophore is caged with a biothiols
reactive 2,4-dinitrobenzenesulfonyl (DNBS) moiety, which blocks the ESIPT process
and quenches the fluorescence. The reaction between probe and biothiols will produce
the HCA fluorophore with large Stokes shift and long-wavelength red fluorescence
emission. The biothiols in solutions and living cells can be conveniently detected with
the probe DNBS-HCA due to its excellent properties such as simple synthetic
procedure, remarkable fluorescence enhancement, good sensitivity, high selectivity
and low cytotoxicity. Benefiting from the AIE characteristic, the probe is also applied
for biothiols detection on indicator papers with RGB color red values, which can be
handily obtained by smartphone app.
Keywords:
Fluorescent probe; Biothiols; Aggregation induced emission; ESIPT; Chalcone
2

1. Introduction
Biological thiol compounds (biothiols), such as glutathione (GSH), Cysteine (Cys)
and Homocystine (Hcy), are closely related to numerous physiological and
pathological processes in living organisms and cells. Due to the redox properties of
the thiol groups, these biothiols play crucial roles in intracellular redox homeostasis,
detoxification, proteins folding, activity regulation of enzymes, cellular signal
transduction, and gene expression, etc [1, 2]. Abnormal concentrations of these
biothiols have been reported to associate with some disease processes such as cancer,
inflammation, cardiovascular diseases, and neurodegenerative diseases [3-5]. Owing
to the significance of these biothiols for living organisms, the detection of these
biothiols is of great importance for the understanding of the biological functions, also
for the diagnosis and treatment of related diseases.
Fluorescent probes are powerful tools for sensitive detection of biothiols in
biological samples. Various different kinds of organic fluorophores have been
employed for fluorescent sensing and imaging of intracellular biothiols with high
temporal-spatial resolution [6-13]. However, most of the ordinary fluorescent probes
for biothiols detection show aggregation-caused quenching (ACQ) effect and
quenched fluorescence in aqueous biological samples. In contrast, some novel
fluorophores (e.g., tetraphenylethene and hexaphenylsilole) with the aggregation
induced emission (AIE) characteristic have been reported to overcome this defect
[14-16]. These AIE fluorophores reveal good features such as higher brightness, lower
background noise and better photostability, which are beneficial for fluorescent
3

imaging of biothiols in living cells [17-24].
Scheme 1. The mechanism of the probe DNBS-HCA response to biothiols.
Herein, we design and synthesis a novel turn-on fluorescent probe DNBS-HCA for
biothiols detection and imaging based on a simple 2′-Hydroxychalcone (HCA)
fluorophore caged by a 2,4-dinitrobenzenesulfonyl (DNBS) moiety acting as
biothiols-reactive fluorescence quencher (Scheme 1). The N, N-dimethylamino unit
and 2'-hydroxyacetophenone unit in the HCA fluorophore are respectively regarded
as electron donor and acceptor (D-A) of the intramolecular charge transfer (ICT)
process [25]. The HCA fluorophore also possesses an excited-state intramolecular
proton transfer (ESIPT) coupled AIE characteristic [26-34]. All these processes
produce the large Stokes shift (>140 nm) and red shift of fluorescence wavelength.
The fluorescence property of the probe DNBS-HCA is totally different from the
HCA fluorophore. The DNBS moiety introduced to cage the 2′-hydroxyl group of
HCA fluorophore can quench the fluorescence through the photo-induced electron
transfer (PET) effect. Moreover, the ESIPT process is also blocked when the hydroxyl
group in 2'-hydroxyacetophenone unit is caged [26, 35, 36]. The reaction between
probe DNBS-HCA and biothiols will produce a 2,4-dinitrothiophenolate (DNTP) and
the HCA product, which gives rise to a turn-on red fluorescence (Scheme 1 and Fig.
4

S1). This reaction mechanism between 2,4-dinitrobenzenesulfonate ester and biothiols
has been verified by many reports [37-48]. The AIE characteristic of the HCA
product promises a remarkable fluorescence enhancement and the feasibility for
biothiols detection in the solid-phase on indicator papers.
2. Experimental section
2.1. Reagents and general methods
All chemical reagents were purchased from commercial companies and used
without further purification. The NMR spectra were carried out on a 400 MHz Bruker
ADVANCE III spectrometer. Mass spectrum was carried out on a Thermo Scientific
LTQ Orbitrap XL FTMS. HPLC test was carried out on a Waters HPLC-1525. The
UV-vis absorption spectra were measured by using an Agilent Cary 60 spectrometer.
Fluorescence spectra were recorded on a ThermoFisher Scientific Lumina
fluorescence spectrometer. Fluorescent imaging of living cells was performed on a
Carl Zeiss LSM800 Confocal Laser Scanning Microscope (CLSM).
2.2. Synthesis of DNBS-HCA
The 2′-hydroxychalcone (HCA) was easily synthesized by a simple one-step
condensation reaction according to the reported method [49].
A
2'-hydroxyacetophenone (4.087 g, 0.03 mol) and 4-dimethylaminobenzaldehyde
(4.467 g, 0.03 mol) were added to a 250 mL round-bottom flask and dissolved by
adding ethanol (50 mL). Then, the KOH (4.208 g, 0.075 mol) was added. The reaction
mixture was reflux at 60 °C for 12 h. After the solution was cooled, 3 M HCl was
added to adjust the pH to 6. The solid precipitation was filtered out and the residual
5

solvent was removed under reduced pressure. The crude product was purified through
silica gel column chromatographic (PE:EtOAc=10:1 as eluent). A red pure solid
1
product was obtained (6.30 g, 78%). H NMR (400 MHz, Chloroform-d): δ 13.26 (s,
1H), 7.95 (dd, J = 8.3, 6.9 Hz, 2H), 7.61 (d, J = 8.9 Hz, 2H), 7.52-7.46 (m, 2H), 7.04
(dd, J = 8.3, 0.8 Hz, 1H), 6.97-6.92 (m, 1H), 6.73 (d, J = 8.9 Hz, 2H), 3.09 (s, 6H).
The HCA (91.5 mg, 0.34 mmol) was added to a 100 mL round bottom flask and
dissolved in 7 mL CH2Cl2. Et3N (100 μL) was added to the reaction in an ice bath,
stirred at 0 °C for 30 min. Then, 2,4-dinitrobenzenesulfonyl chloride (183.5 mg, 0.68
mmol) was added, and the reaction was conducted at room temperature for 8 h. The
solvent was removed under reduced pressure. The crude product was purified by
silica gel column chromatography (PE:EtOAc=5:1 as eluent). A brown solid product
1
DNBS-HCA was obtained (110 mg, 64%). H NMR (400 MHz, Chloroform-d): δ
8.34-8.29 (m, 2H), 8.19-8.13 (m, 1H), 7.68-7.58 (m, 2H), 7.54-7.45 (m, 2H),
7.26-7.16 (m, 3H), 6.71 (d, J = 15.6 Hz, 1H), 6.59 (d, J = 8.8 Hz, 2H), 3.08 (s, 6H).
13C NMR (100 MHz, DMSO-d6) δ 189.52, 152.64, 151.32, 148.29, 146.87, 145.93,
134.51, 133.75, 133.06, 131.85, 131.16, 130.86, 128.90, 127.92, 123.69, 121.37,
121.22, 120.01, 112.15, 39.91. HRMS (ESI) m/z: calcd [M+H]+ for C23H20N3O8S
498.0971, found 498.0959.
2.3. Spectra properties of DNBS-HCA
The stock solutions of DNBS-HCA (10 mM) and HCA (10 mM) were prepared in
DMSO. The test solutions of DNBS-HCA (40 μM) and HCA (40 μM) were prepared
by diluting the stock solution with DMSO-HEPES buffer (v/v = 3:7, 50 mM, pH =
6

7.4).
The absorption and fluorescence spectra of DNBS-HCA with or without different
biothiols (400 μM, 10 equiv) were measured. The fluorescence response of
DNBS-HCA with different concentration of biothiols were recorded after incubating
with these biothiols for 60 min at room temperature.
The probe DNBS-HCA with excess Cys was prepared in MeOH/H2O (v/v = 5/5)
1
solution for HPLC and mass spectra test. The sample used for H NMR test was
prepared in DMSO/H2O (v/v = 3/7) solution and extracted with the ethyl acetate. The
1
extract from ethyl acetate phase was dissolved in deuterium DMSO and used for H
NMR test.
The limits of detection (LOD) for different biothiols were determined by using the
3δ/k, where δ is the standard deviation (SD) of the blank sample solution without any
biothiols and k is the slope of the fitted curve of fluorescence intensity with biothiols
concentration. The time-dependent fluorescence responses of DNBS-HCA were
recorded with the addition of excess biothiols (400 μM, 10 equiv).
The selectivity of DNBS-HCA for biothiols (Cys, GSH and Hcy) was evaluated by
the fluorescence response with the addition of excess biological species (400 μM, 10
equiv) including different amino acids (Gly, Tyr, Leu, Gln, Glu, Arg, Thr, Lys), metal
ions (K+, Na+, Mg2+, Cu2+, Fe2+), and protein BSA (1 mg/mL). The biothiols (400 μM)
pretreated with equivalent thiol scavenger N-ethyl-maleimide (NEM, 400 μM) were
also detected.
2.4. Detection of biothiols on indicator papers
7

The indicator papers of probe DNBS-HCA for biothiols detection were prepared
with filter papers (φ=0.5 cm) by immersing into solution of DNBS-HCA (1 mM)
dissolved in acetonitrile and then drying in atmosphere. The solutions (10 μL) of
biothiols with different concentrations in DMSO-HEPES buffer (v/v = 3:7, 50 mM,
pH = 7.4) were dropped to the indicator papers and incubated for 30 min. The
photograph of the fluorescence on the indicator papers was taken by a smartphone
camera under a 365 nm UV light. The relative intensities of the red fluorescence were
quantified with the red values of RGB color obtained by a smartphone application
(Color recognizer) as the previous report [50].
2.5. Cell culture and fluorescent imaging
Human prostate cancer PC3 cells and human lung adenocarcinoma A549 cells were
maintained in a CO2 incubator (ESCO) at 37°C with humidified atmosphere of 5%
CO2. The cells were cultured and prepared for fluorescent imaging as previous
described [51], which were cultured in Dulbecco’s Modified Eagle’s Medium
(DMEM, Hyclone) supplemented with 10% fetal bovine serum (FBS, Gibco),
penicillin (100 U/mL) and streptomycin (100 μg/mL). The cells used for fluorescent
imaging were cultured in glass-bottom dishes (φ=20 mm) for 24 h, and then treated
with or without 0.5 mM thiol scavenger NEM in PBS for 30 min at 37°C. After
washed with PBS (3×1 mL), the cells were stained with DNBS-HCA (10 μM in PBS)
at 37°C for 30 min. The cells were then washed with PBS (3×1 mL) and imaged using
the CLSM with a ×63 objective lens. The excitation wavelength was 488 nm. The
emission wavelength was 560-700 nm for red channel and 500-560 nm for green
8

channel. The average fluorescent intensity in fluorescent imaging was measured by
the software Image J.
2.6. Cytotoxicity of DNBS-HCA
The cell counting kit-8 (CCK-8, Beyotime Biotechnology) was used to assess the
cytotoxicity of DNBS-HCA. The A549 cells were seeded on 96-well cell culture
plates (3×103 cells/well, 100 μL) and cultured for 24 h. Then the probe DNBS-HCA
dissolved in 10 μL medium was added into the wells with the final concentration from
0 to 16 μM and incubated for 24 h. The CCK-8 solution (10 μL) was then added and
incubated for 1 h. The optical density (OD) at 450 nm was recorded by a multimode
microplate reader (Varioskan Flash, Thermo Scientific). The cell viabilities were
obtained from the OD450 nm ratio between DNBS-HCA treated groups and control
group.
3. Results and discussion
3.1. Design and synthesis of the probe DNBS-HCA
The synthetic procedure of DNBS-HCA is very simple and convenient (Scheme 2).
The HCA fluorophore was readily synthesized following the literature method [26, 36,
49] through a simple one-step Claisen-Schmidt condensation reaction between
2'-hydroxyacetophenone and 4-dimethylaminobenzaldehyde with high yield 78%. The
probe DNBS-HCA was then prepared with the HCA fluorophore and
2,4-dinitrobenzenesulfonyl chloride in 64% yield. The structure of DNBS-HCA was
characterized by 1H NMR, 13C NMR, and HRMS (Fig. S2).
9

Scheme 2. Synthetic route of the probe DNBS-HCA.
3.2. Spectra properties of the HCA fluorophore
HCA fluorophore is the prospective product of the reaction between DNBS-HCA
and biothiols (Scheme 1). It has been reported that the HCA fluorophore possesses an
ESIPT active AIE phenomenon [26-31]. The fluorescent spectra of this fluorophore in
different solvents are shown in Fig. S3. It displays weakly green fluorescence in some
organic solvents. In contrast, it exhibits stronger, red fluorescence in the aqueous
solution. The mixed solvent system (water/THF) is used to verify the AIE
characteristic. The fluorescence shows gradually increased intensity and red-shifted
wavelength following the rise of the water content (Fig. S4). The UV-vis absorption
spectrum and fluorescence spectrum of HCA in mixed DMSO-HEPES buffer (v/v =
3:7, 50 mM, pH = 7.4) are shown in Fig. S5. The AIE property of HCA was further
performed in mixed DMSO/HEPES buffer. As shown in Fig. S6, the
aggregation-induced red emission was enhanced with the increase of water proportion.
The mixed DMSO/HEPES buffer (v/v = 3:7, 50 mM, pH = 7.4) is a critical fraction
for HCA at the concentration of 40 μΜ. When 100 μΜ HCA was dissolved in this
buffer, a significant aggregation-induced red emission will produce. We also confirm
the particle size of the aggregated HCA fluorophore in HEPES buffer fraction
10

exceeding 70%. The dynamic light scattering (DLS) test was used to observe the
particle size. As shown in Fig. S7, the particle size is increased with the rise of
HEPES buffer fraction. The large Stokes shift (about 140 nm) of the HCA
fluorophore can reduce the self-absorption and quenching, which is remarkable for
fluorescence detection and imaging.
The fluorescence property of HCA product in different pH solutions were
investigated. As shown in Fig. S8, the fluorescent intensity of HCA displays good
stability in the range of pH 3-9 with 30% DMSO. While in the strong base solution of
pH 11, the fluorescence spectrum of HCA shows a distinct blue-shift and a decrease
of fluorescence intensity.
3.3. Spectra properties of DNBS-HCA for biothiols detection
The absorption spectra of DNBS-HCA for biothiols detection were firstly
measured. As shown in Fig. 1a, the probe DNBS-HCA displays an absorption peak at
440 nm with a molar extinction coefficient (ε = 1.8 × 104 M−1cm−1) in DMSO-HEPES
buffer (v/v = 3:7, 50 mM, pH = 7.4). After reacting with Cys, the absorption peak
reveals a weak red-shift to 460 nm and slightly enhanced intensity with a molar
extinction coefficient (ε = 2.6 × 104 M−1cm−1). Meanwhile, the absorption appears at
300-400 nm after reacting with Cys is attributed to the DNTP product [17]. The HCA
fluorophore was also brought into the measurement as a control, because it is the
expected fluorescence product of reaction between DNBS-HCA and biothiols. Indeed,
the absorption spectra of DNBS-HCA with Cys is exactly similar to that of the HCA
fluorophore with a molar extinction coefficient (ε = 2.8 × 104 M−1cm−1). All the
11

spectral changes are in accord with the predictable mechanism of a
2,4-dinitrobenzenesulfonate ester for biothiols detection (Scheme 1 and Fig. S1).
Some other experiments such as 1H NMR, mass spectra and HPLC were carried out to
verify the mechanism and the product (Fig. S9, S10 and S11). The 1H NMR spectrum
of DNBS-HCA with excess Cys is totally same as that of HCA. The HRMS for HCA
product [M+H]+ was calcd to 268.1338, and found 268.1336. The retention times of
chromatograph peak in HPLC directly reveals the produce of HCA and the decrease
of DNBS-HCA. All these results confirm the product is the HCA fluorophore.
Fig. 1. The spectra properties of probe DNBS-HCA (40 μM) with or without biothiols. (a) The
absorption spectra of HCA, DNBS-HCA with or without Cys (400 μM). (b) The fluorescence
spectra of HCA, DNBS-HCA with (400 μM) or without different biothiols (GSH, Cys and Hcy).
The fluorescence spectra of HCA, DNBS-HCA and DNBS-HCA with different
12

biothiols (GSH, Cys and Hcy) are shown in Fig. 1b. The probe DNBS-HCA (40 μM)
in DMSO-HEPES buffer (v/v = 3:7, 50 mM, pH = 7.4) shows almost no fluorescence,
which is attributed to the quench effect of the DNBS moiety. After the addition of
excess biothiols (10 eq), a remarkable turn-on fluorescence peak at 600 nm is
observed. It implies the remove of the biothiols-reactive DNBS moiety. The emission
intensity at 600 nm shows 94-fold, 92-fold and 51-fold enhancement for addition of
GSH, Cys and Hcy, respectively. The fluorescence intensity of DNBS-HCA with
excess GSH is close to that of the HCA fluorophore. These results indicate the
produce of HCA product with a remarkable turn-on fluorescence from reaction
between DNBS-HCA and biothiols.
3.4. Quantitative detection of biothiols with DNBS-HCA
The quantitative analysis of DNBS-HCA for biothiols detection was measured by
gradual addition of different biothiols (GSH, Cys or Hcy). As shown in Fig. 2a, the
fluorescence spectra reveal significantly and gradually increase with the addition of
various amounts of GSH. The fluorescence of DNBS-HCA with various amounts of
Cys or Hcy reveals a similar trend (Fig. S12). The fluorescence intensity at 600 nm
was calculated to clarify the sensitivity of the reaction between DNBS-HCA and
biothiols. In Fig. 2b, the fluorescence intensity at 600 nm is enhanced continuously
with the increase of the biothiols concentration until it reaches a maximum plateau
with a few equiv (1-5 equiv) of biothiols.
In the low concentration range of biothiols, the fluorescence intensity at 600 nm
shows good linear correlation with the biothiols concentration (Fig. 2c). The linear
13

correlation coefficient for GSH, Cys and Hcy are 0.977, 0.977 and 0.988, respectively.
The limits of detection (LOD) for GSH, Cys and Hcy are determined to be 0.227,
0.283 and 1.826 μM according to the 3δ/slope. These results demonstrate
DNBS-HCA has good sensitivity for biothiols sensing.
Fig. 2. Fluorescence of DNBS-HCA (40 μM) upon addition of different concentrations of
biothiols (0-400 μM). (a) The fluorescence spectra of DNBS-HCA (40 μM) with different
concentrations of GSH. (b) The relationship between fluorescence intensity of DNBS-HCA at 600
nm and biothiols concentrations. (c) The fluorescence intensity of DNBS-HCA at 600 nm as a
function of biothiols in the low concentration range.
3.5. Time-dependent fluorescence responses of DNBS-HCA for biothiols
14

The time-dependent fluorescence responses of DNBS-HCA for biothiols were
determined. As shown in Fig. 3a and Fig. S13, the fluorescence band at 600 nm
displays gradually increasement with excess biothiols (10 equiv GSH, Cys or Hcy).
The fluorescence intensity at 600 nm was also collected to evaluate the response
efficiency over time with biothiols. The fluorescence intensity reaches its maximum
value in less than 15 minutes (approximately 3 min for Cys, and 15 min for GSH,
Hcy). The kinetics process of biothiols response with DNBS-HCA reveals the fast
reactions between them.
Fig. 3. Time-dependent Fluorescence of DNBS-HCA (40 μM) upon addition of different biothiols
(10 eq). (a) Time-course fluorescence spectra of DNBS-HCA (40 μM) with GSH (400 μM, 10
equiv). (b) Fluorescence intensity changes at 600 nm of DNBS-HCA (40 μM) in the presence of
GSH, Cys, or Hcy (400 μM).
The effect of water volume on the biothiol-sensing kinetics process was evaluated.
15

The fluorescent intensity at 600 nm was collected to reveal the kinetics process, which
was shown in Fig. S14. The results demonstrate that the increase of water volume
from 70% to 100% will decrease the reaction rate. The kinetics process of probe
DNBS-HCA in different pH solutions (with 30% DMSO) was also verified. As
shown in Fig. S15, The acidic pH 5 causes the decrease of reaction rate, while the
alkaline pH 9 promotes the detect reaction.
3.6. Selectivity of DNBS-HCA for biothiols
The selectivity of DNBS-HCA for biothiols (Cys, GSH, Hcy) was evaluated by
comparison with various biological species including amino acids (Gly, Tyr, Leu, Gln,
Glu, Arg, Thr, Lys), representative metal ions (K+, Na+, Mg2+, Cu2+, Fe2+) and protein
BSA. The fluorescence intensity of DNBS-HCA with all these species shows almost
no change compared with that of the probe. The remarkable enhancement of the
fluorescence intensity with Cys, GSH or Hcy indicates the high selectivity of
DNBS-HCA for biothiols over these species (Fig. 4). Noteworthily, the fluorescence
of DNBS-HCA with protein BSA containing some disulfide bonds and free thiol has
also no change, indicating the probe DNBS-HCA will not react with non-protein
thiols.
The selectivity of DNBS-HCA for biothiols sensing in solutions was also verified
by using the thiol scavenger NEM (Fig. 4). DNBS-HCA reveals very weak
fluorescence enhancement with Cys and GSH pretreated by equivalent NEM. The
thiol of Cys and GSH is almost blocked by the scavenger NEM, which results in the
negligible fluorescence changes.
16

Fig. 4. Relative fluorescence intensity of DNBS-HCA (40 μM) at 600 nm with different
biological species (400 μM, 10 equiv) including amino acids (Gly, Tyr, Leu, Gln, Glu, Arg, Thr,
Lys), metal ions (K+, Na+, Mg2+, Cu2+, Fe2+), protein BSA (1 mg/mL), and biothiols (400 μM)
pretreated with equivalent NEM.
3.7. Detection of biothiols on indicator papers with DNBS-HCA
In consideration of the AIE characteristic of the HCA product from the detection
reaction, the availability of the probe DNBS-HCA for biothiols detection on indicator
papers was carried out. Filter papers deposited with DNBS-HCA were used for
biothiols detection in solid phase. The solutions of biothiols with different
concentrations were dropped onto the indicator papers. The photograph of the turn-on
red fluorescence on the indicator papers with the addition of Cys and GSH was taken
under 365 nm UV light by a smartphone camera (Fig. 5a and Fig. S16a). The red
color of fluorescence on indicator papers shows remarkable enhancement following
the increase of biothiols concentration.
17

Fig. 5. Photograph of fluorescence and intensities on indicator papers with DNBS-HCA for Cys
detection. (a) Photograph of fluorescence on indicator papers with DNBS-HCA for Cys detection.
(b) Plot of RGB red values of fluorescence on indicator papers with Cys concentration. Inset:
Linear range of RGB red values of fluorescence with Cys concentration.
The relative intensity of the red fluorescence on indicator papers was calculated to
obtain the quantitative relation with the concentration of biothiols. The red values of
the RGB color in the photographs were also obtained by a convenient smartphone
application (Color recognizer). As shown in Fig. 5b and Fig. S16b, the red values of
the RGB color continuously increase until it reaches a maximum plateau. Particularly,
the red values have a good linear relationship (R2 = 0.990) with the Cys concentration
in the range of 0.1-1 mM. These results demonstrate the feasibility and sensitivity for
quantitative detection of biothiols with DNBS-HCA on indicator papers.
3.8. Cytotoxicity and fluorescent imaging in living cells
The cytotoxicity of the probe DNBS-HCA on living cells was tested by the CCK-8
assay. A549 cells were used and incubated with different concentrations of probe
(0-16 μM) for 24 h. As shown in Fig. 6, the cell viability is greater than 95%, even
18

when the cells were incubated with probe DNBS-HCA (16 μM) for 24 h. It indicates
the low cytotoxicity of the probe on A549 cells.
Fig. 6. The cell viability of DNBS-HCA on A549 cells for 24 h with CCK-8 assay.
Finally, we examined the capability of DNBS-HCA to visualize endogenous
biothiols in living cells. The fluorescence imaging of A549 cells stained with probe
DNBS-HCA for 30 min is shown in Fig. S17, the strong fluorescence in the red
channel (560-700 nm) and weak fluorescence in the green channel (500-560 nm) can
be observed. The red fluorescence should be attributed to the aggregation state of
HCA molecule produced from the reaction between DNBS-HCA and biothiols in
cells.
In addition, the results described above demonstrate that the increase of water
volume will decrease the reaction rate. This result seems inconsistent with the rapid
fluorescence response in living cells, which is totally a water environment. We
conjecture that is related to the cellular contents such as proteins. The bovine serum
albumin (BSA) in solutions was used to verify this hypothesis. As shown in Fig. S18a,
the fluorescence of probe DNBS-HCA (40 μM) is almost no changes even with
excess Cys (10 eq) or BSA (1mg/mL) in pure water, respectively. However, the
19

fluorescence shows remarkable enhancement with Cys (10 eq) in BSA (1mg/mL)
solution. The reason should be attributed to the promotion of the dispersity of the
probe in aqueous solution with the proteins. The biothiol-sensing dynamic curves in
BSA solutions were also measured (Fig. S18b). The increase of BSA concentration
from 1 to 10 mg/mL will facilitates the reaction rate. So the rapid fluorescence
response in living cells is reasonable.
In order to validate the red fluorescence in living cells comes from the biothiols
induced probe response, control experiments of cells pretreated with thiol scavenger
were used to carry out the fluorescence imaging. In Fig. 7, PC3 cells and A549 cells
stained with the probe show remarkable fluorescence. In contrast, the cells pretreated
with the thiol scavenger NEM (0.5 mM for 30 min) show weak fluorescence. When
the biothiols in living cells are eliminated by the scavenger NEM, no more biothiols
can react with the probe DNBS-HCA to produce the fluorescent HCA. Meanwhile,
the fluorescence in blank control without probe is very weak (Fig. S19a, f). The effect
of NEM concentration on biothiol imaging was also studied with two different
concentrations (0.1 and 0.5 mM). The results indicate that the fluorescence is
decreased with the increasing of NEM concentration (Fig. S19b-d, and f). In addition,
cells pretreated with NEM were further incubated with Cys to use as a positive control.
A significant increase of fluorescence was obtained with the addition of Cys (Fig.
S19e, f). These results demonstrate the availability and specificity of DNBS-HCA for
biothiols detection in living cells.
20

Fig. 7. (a) Fluorescent imaging of biothiols in living cells with probe DNBS-HCA in red channel.
The second row: Cells pretreated with NEM (0.5 mM). BF: bright field imaging. λex = 488 nm,
λem = 560-700 nm. Scale bar = 10 μm. (b) The average fluorescent intensity in cells. Error bars
represent standard deviation. Significance was determined using the T test (***, P < 0.001).
4. Conclusion
In summary, we have developed a novel turn-on fluorescent probe DNBS-HCA for
biothiols detection based on the 2′-Hydroxychalcone (HCA) fluorophore possessing
an
ESIPT
coupled
AIE
characteristic.
The
biothiols
reactive
2,4-dinitrobenzenesulfonyl (DNBS) moiety of the probe DNBS-HCA is employed to
block the ESIPT process and quench the fluorescence through PET process. The
response reaction of probe DNBS-HCA with biothiols will produce the HCA
fluorophore with large Stokes shift (140 nm) and long-wavelength red fluorescence
emission. The probe reveals remarkable fluorescence enhancement of 51- to 94-folds
and fast response time of 3 to 15 mins with different biothiols. The biothiols in
solution and living cells can be conveniently detected with the probe DNBS-HCA due
to its excellent properties such as simple synthetic procedure, low cytotoxicity, good
sensitivity and high selectivity. In addition, the AIE characteristic of the HCA product
21

also provides strategy for biothiols detection on indicator papers with RGB color red
values, which can be handily obtained by smartphone application.
Acknowledgements
This work is supported by National Natural Science Foundation of China (21708023),
China Postdoctoral Science Foundation (2019M653851XB), Natural Science Basic
Research Plan in Shaanxi Province of China (2019JQ-061, 2019JQ-789).
22

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