FRET-based dual channel fluorescent probe for detecting endogenous/exogenous H2O2/H2S formation through multicolor images

Article abstract of DOI:10.1016/j.jphotobiol.2018.12.016

We have developed a FRET-based fluorescent probe (PHS1) as a combination of two different fluorophores (coumarin and naphthalimide); which can detect both exogenous and endogenous H₂S and H₂O₂ in live cells through multicolor images. The precise overlap between UV-absorption of naphthalimide and the emission band of coumarin in probe PHS1 allows the acquisition of the self-calibrated information of dual analytes through FRET-based imaging. The UV–Vis absorption (λ_abs 390 nm) and fluorescence emission (λ_em 460 nm) of probe PHS1 in the presence of H₂O₂ are increased ∽35- fold and ∽15-fold respectively. It also allows the estimation of the levels of H₂S through enhancement of emission intensity at 550 nm. The probe PHS1 exhibits high stability against various analytes, including various pH (4–9.5). The cell viability assay data indicate that the probe is not harmful to the cancer cells. The nontoxic nature of the probe PHS1 encourages application for cancer cell labeling. The probe PHS1 can detect the level of endogenous H₂O₂, H₂S, and H₂O₂/H₂S in cancer cells through blue, green and FRET-based green channel imaging. PHS1 is a unique probe, has potential application for diagnosing cancer by providing information on the level of dual analytes (H₂S, H₂O₂) in cancer cells.

Full text of DOI:10.1016/j.jphotobiol.2018.12.016

Journal of Photochemistry & Photobiology, B: Biology 191 (2019) 99–106
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
FRET-based dual channel fluorescent probe for detecting endogenous/
exogenous H O /H S formation through multicolor images
2
2
2
a,1
b,1
a
a
Nithya Velusamy , Natesan Thirumalaivasan , Kondapa Naidu Bobba , Arup Podder ,
b,⁎
a,c,⁎⁎
Shu-Pao Wu , Sankarprasad Bhuniya
a
b
c
Amrita Centre for Industrial Research & Innovation, Amrita School of engineering, Coimbatore Amrita University, 64112, India
Department of Applied Chemistry, National Chiao Tung University, 1001 Ta Hsueh Road, Hsinchu 300, Taiwan
Department of Chemical Engineering & Materials Science, Amrita School of Engineering, Coimbatore, Amrita Vishwa Vidyapeetham, Amrita University, 641112, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
We have developed a FRET-based fluorescent probe (PHS1) as a combination of two different fluorophores
FRET- based fluorescent probe
Coumarin
(coumarin and naphthalimide); which can detect both exogenous and endogenous H S and H O in live cells
2
2 2
through multicolor images. The precise overlap between UV-absorption of naphthalimide and the emission band
of coumarin in probe PHS1 allows the acquisition of the self-calibrated information of dual analytes through
FRET-based imaging. The UV–Vis absorption (λabs 390 nm) and fluorescence emission (λem 460 nm) of probe
Naphthalimide
Endogenous H
2
2
O
2
Endogenous H
S
PHS1 in the presence of H
2
O are increased ∽35- fold and ∽15-fold respectively. It also allows the estimation of
2
the levels of H S through enhancement of emission intensity at 550 nm. The probe PHS1 exhibits high stability
2
against various analytes, including various pH (4–9.5). The cell viability assay data indicate that the probe is not
harmful to the cancer cells. The nontoxic nature of the probe PHS1 encourages application for cancer cell
labeling. The probe PHS1 can detect the level of endogenous H
2
O
2
, H
2
S, and H
2
O
2
/H S in cancer cells through
2
blue, green and FRET-based green channel imaging. PHS1 is a unique probe, has potential application for di-
agnosing cancer by providing information on the level of dual analytes (H S, H ) in cancer cells.
2
O
2 2
1
. Introduction
Hydrogen peroxide (H
significant role in several physiological processes, including cardio-
vascular, gastrointestinal, immune defense, and nervous systems
[11,12]. Indeed, it supports tumor growth via stimulating the angio-
genesis, regulating mitochondrial bioenergetics, stimulation of cell
cycle progression and anti-apoptosis function [13]. Apart from cancer,
2
O
2
) and hydrogen sulfide (H S) are en-
2
dogenously produced small reactive molecular species. Those have a
significant role in physiological and pathological consequences in living
organisms [1–4]. In physiological condition, they play a pivotal role in
redox signaling pathway [5]. Depending on their concentrations and
locations they exhibit pro- or anti-apoptotic activities and behave like a
irregular production of endogenous H S is associated with numerous
2
diseases such as Alzheimer's disease, Down syndrome, diabetes, and
liver cirrhosis [14–17]. The enzymes cystathionine-β-synthase (CBS)
and cystathionine-γ-lyase (CSE) are mainly responsible for over-
double-edged sword in cancer prognosis [6,7]. H
2
O is a crucial ROS
2
produced in the mitochondrial respiratory chain cascade. It participates
in various physiological processes, namely, cell proliferation and dif-
ferentiation, and also in hypoxia signal transduction [8]. In cancer cells,
production of H S in cancer cells [18]. Moreover, CBS induces oxidative
2
stress. Taking advantage of fluorescence modality, several fluorescent
probes have been developed to expose and visualize various biological
events through fluorescence imaging in vitro as well as in vivo [19–27].
In the present decade, several fluorescent probes have visualized both
the anaerobic glycolysis process predominantly produces superoxide
−
anion (O
2
); which rapidly reacts with appropriate superoxide dis-
mutase to generate H
2
O
2
. This ROS deactivates functions of tumor
endogenous and exogenous H
2
O
2
[28–35] and H S [36–45] formation
2
suppressor gene p53 and activates several oncogenic pathways such as
in vitro and within live-specimens. Chang and his co-worker observed
that NADPH-oxidase derived H stimulate endogenous H S forma-
tion in human umbilical vein endothelial cells [46]. Thus, we are
RAS, MYC, and AKT to accelerate tumor progression [9,10]. On the
2
O
2
2
other hand, hydrogen sulfide (H S) as the third gasotransmitter plays a
2
⁎
Corresponding author.
⁎
⁎
Corresponding author at: Amrita Centre for Industrial Research & Innovation, Amrita School of engineering, Coimbatore Amrita University, 64112, India.
E-mail addresses: spwu@mail.nctu.edu.tw (S.-P. Wu), b_sankarprasad@cb.amrita.edu (S. Bhuniya).
1
These authors equally contributed to this study.
https://doi.org/10.1016/j.jphotobiol.2018.12.016
Received 9 October 2018; Received in revised form 18 December 2018; Accepted 19 December 2018
Available online 26 December 2018
1011-1344/ © 2018 Published by Elsevier B.V.

N. Velusamy et al.
Journal of Photochemistry & Photobiology, B: Biology 191 (2019) 99–106
interested in developing FRET-based ratiometric fluorescent probes to
(d, 2H, J = 6.0 Hz), 4.297 (q, 2H, J = 5.6 Hz), 2.496 (s, 3H), 1.315 (s,
1
3
detect H
2
O
2
, H
2
S, and the combination of them (H
2
O
2
/H
2
S) in cancer
15 Hz). C NMR (100 MHz, DMSO-d ). 12.82, 21.50, 97.54, 111.21,
6
cells; the early information on H
2
O
2
/H
2
S can help in diagnosing cancer
117.62, 125.02, 126.98, 127.09, 127.44, 128.95, 129.48, 130.46,
135.91, 136.43, 145.52, 152.69, 152.87, 169.26, 172.42. ESI- HRMS
m/z (M + H+): calcd. 359.1588, found 359.1666.
and open a new research avenue for tracking of cancer cells. Moreover,
this dual sensing strategy simultaneously can provide information on
two different analytes (H
2
O
2
/H S), and their intracellular location
2
through dual-channel imaging.
2.2.4. Synthesis of 4
To compound 3 (600 mg, 2.30 mmol) in aqueous THF (10.0 mL),
LiOH (320 mg) was added and stirred for 1 h. After completion of re-
action, pH was adjusted to ∽5 using HCl (2.0 N) and then diluted with
ethyl acetate. The combined organic layers were washed with water
and brine. It was then dried over anhydrous sodium sulfate and eva-
2
. Experimental Procedures
2.1. General Information on Materials, Methods and Instrumentations
Diethylmalonate (Avra, India), 2,4-dihydroxy-3-methylbenzalde-
porated under reduced pressure to afford compound 4 as pale yellow
1
hyde (Alfa Aesar, India), piperidine (Avra, India), bromo-1,8-naphthalic
anhydride (TCI, Japan), ethanol (Changshu Yangyuvan Chemical,
China), ethylenediamine (Sigma Aldrich, India), sodium azide (Loba
Chem, India), DMSO (Loba Chem, India), lithium hydroxide (Himedia,
India), N-Phenyl-bis(trifluoromethanesulfonimide) (Avra, India), bis
solid (340 mg, 69.16%). H NMR (400 MHz, DMSO-d
6
): δ 8.655 (s,
1H),7.281 (t, 1H, J = 6.01 Hz), 6.909 (d, 1H, J = 6.80 Hz), 2.365 (s,
1
3
3H), 1.320 (s, 12H). C NMR (100 MHz, DMSO-d )0.14.32, 25.42,
6
84.56, 113.37, 124.84, 127.04, 131.49, 135.75, 148.49, 153.25,
158.37, 164.74. ESI- HRMS m/z (M + Na+): calcd. 353.1165, found
353.1162.
(
pinacolato)diboron (Avra, India), 1,1′-Bis(diphenylphosphino) ferro-
cene dichloropalladium (II) complex with dichloromethane (Avra,
India),
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
hydro-
2.2.5. Synthesis of A
chloride (Avra, India), hydroxybenzotriazole hydrate (Himedia, India),
sodium carbonate (Himedia, India), THF (Merck, India), DMF (Avra,
India), potassium acetate (Himedia, India), toluene (Avra, India), 4- and
sodium sulfate (Loba Chem, India) were procured from commercial
sources and used as such. In column chromatography silica gel
The compound A was synthesized according to the literature [48].
2.2.6. Synthesis of PHS1
HOBT (76 mg, 0.545 mmol) and EDCI (110 mg, 0.545 mmol) were
added to a solution of 4 (180 mg, 0.542 mmol) in DMF and stirred for
15 min at RT. Then, A (184 mg, 0.654 mmol) was added to the reaction
mixture. The reaction mixture was stirred overnight 50 °C and then
poured into water (10 mL). The precipitated was filtered, washed with
water, and dried under a vacuum; finally, it was purified by column
(
100–200 mesh, Loba Chem) was used as the stationary phase.
Shimadzu UV-1800 spectrophotometer was used for recording UV–Vis.
spectra. NMR spectra were taken on a 400 MHz spectrometer (Bruker,
Germany). Mass spectra were collected on IonSpec HiResESI mass
spectrometer.
chromatography (DCM/MeOH: 15/1) to get 150 mg of compound PHS1
1
as a yellow solid. Yield: 46.38%. H NMR (DMSO-d
6
): 9.04 (s, 1H),
2.2. Synthesis of PHS1
8.825 (d, 1H, J = 12.00 Hz), 8.639 (m, 2H), 8.435 (dd, 1H), 7.714 (m,
2
H), 7.449 (m, 3H), 4.519 (t, 2H, J = 5.60 Hz), 3.888 (q, 2H), 2.653 (s,
1
3
2
.2.1. Synthesis of 1
2H), 2.468 (s, 1H), 1.371 (s, 12H). C NMR (100 MHz, DMSO-d )
6
The compound 1 was prepared as per the previous report [47].
0.14.84, 18.88, 24.61, 24.96, 27.22, 30.12, 71.16, 73.54, 84.12,
115.99, 118.12, 118.68, 122.42, 123.61, 124.69, 125.09, 127.33,
128.33, 128.70, 131.50, 131.73, 135.08, 142.82, 147.58, 152.18,
159.88, 161.69, 162.33, 163.15, 163.58, 179.28. ESI- HRMS m/z
(M + H+): calcd. 594.2082, found 594.2119, (M + Na+): calcd.
616.1979, found 616.2106.
Diethyl malonate (1.263 g, 7.892 mmol) and piperidine (1.680 g,
1
9.731 mmol) were added to a solution of 2, 4-dihydroxy-3-methyl
benzaldehyde (1.0 g, 6.577 mmol) in ethanol (20 mL). The reaction was
continued for 12 h. Next, ethanol was evaporated. The residue was
dissolved in HCl (2 N) and extracted with ethyl acetate. The organic
layer was washed with water, brine and dried over anhydrous sodium
sulfate. Then, it was concentrated under reduced pressure to get com-
pound 1 as pale pink colour solid (1.25 g, 76.64%).
2.3. UV/Vis and Fluorescence Spectroscopy
All fluorescence and UV–Vis. spectra were obtained with RF- 6000
FL spectrometer with a 1 cm standard quartz cell and UV-1800 spec-
2
.2.2. Synthesis of 2
Compound 2 was prepared according to reported article [47]. The
trophotometer, respectively. Sodium sulfide (Na S) was used as the
2
compound 1 (400 mg, 1.6125 mmol) was dissolved in DMF, N-phenyl-
source of H
various analytes (HOCl, H
FeSO , NO, KCl, CaCl , GSH, ascorbic acid (AA), cysteine, folic Acid,
histidine, lysine, NaOCl, K S were prepared in double distilled water.
2
S and H
2
O
2
was used directly. Stock solutions (600 μM) of
bis (trifluoromethyl sulphonamide) (1.1513 g, 3.22 mmol) and Na
2
CO
3
2
O
2
, NaNO , Cu(OAc) , Zn(OAc) , Na O ,
2
2
2
2
S
2
3
(
0.854 g, 8.062 mmol) were added and stirred overnight at rt. Then cold
4
2
water was added and stirred for another 5 min to get a solid precipitate.
The solid was filtered, dissolved in DCM and dried over anhydrous
sodium sulfate. The crude compound was subjected to column chro-
matography to obtain 2 as a pink solid (0.375 g, 61.20%).
2
5
The stock solution of probe PHS1 (40 μM) was prepared in PBS buffer
(pH = 7.4) with 1% DMSO. Excitation was effected at 400 nm and
450 nm with excitation and emission slit widths as 5 nm each. The
fluorescence experiments (solution test) of PHS1 (10.0 μM) were re-
2
.2.3. Synthesis of 3
corded in the presence of increasing concentrations of H
2
O (0–25 eq.)
2
To a solution of 2 (2 g, 5.2629 mmol) in toluene (15 mL), bis(pi-
and Na S (0–25 eq.) in HEPES buffer (pH = 7.4) with 1% DMSO. PHS1
2
nacol)diboron (1.736 g, 6.8412 mmol) and potassium acetate (1.547 g,
was incubated with Na
2
S and H
2
O for 30 min at 37 °C.
2
1
5.789 mmol) were added and purged with nitrogen gas for 15–20 min.
Then, Pd(dppf)Cl
2
(1.289 g, 1.578 mmol) was added to the solution;
2.4. Cytotoxicity Assay
further, purged with nitrogen gas for another 10 min. Following this,
the reaction mixture was stirred at 110 °C for 2 h. The toluene was
evaporated and compound was extracted with ethyl acetate. The or-
ganic layer was dried over anhydrous sodium sulfate. Finally, crude
product was purified by silica gel column chromatography to afford a
By using methyl thiazolyl tetrazolium (MTT) assay, cellular cyto-
toxicity of PHS1 in HT-29 cells was estimated. Several concentrations
(0–50 μM) of PHS1 were added to the wells of the HT-29 cells grown in
a 96-well cell culture plate. Then the cells were maintained under 5%
pale pink solid. 1H NMR (400 MHz, DMSO-d
CO at 37 °C under for 24 h. Subsequently, to each well 10 mL MTT
6
): δ 8.704 (s, 1H), 7.576
2
100

N. Velusamy et al.
Journal of Photochemistry & Photobiology, B: Biology 191 (2019) 99–106
Scheme 1. Synthesis of probe PHS1.
−
1
(
5 mg mL ) was added and incubated at 37 °C under 5% CO
2
for 4 h.
with the Leica TCS SP5 X AOBS Confocal Fluorescence Microscope
(Germany). The excitation and emission wavelength were set at
410 nm/420–470 nm respectively.
Afterward, the MTT solution was removed and the yellow precipitates
(
formazan) were dissolved in 200 mL DMSO and 25 mL sorensen's
glycine buffer (0.1 M glycine and 0.1 M NaCl). The absorbance of each
well at 570 nm was measured using Mullikan GO microplate reader. By
using the following equation the cell viability was calculated: Cell
viability (%) = (Mean of absorbance values of treatment group)/(Mean
of absorbance values of control group).
2.7. FRET Imaging of Exogenous H
2
O
2
and H S in HeLa Cells
2
As mentioned above, the cells were cultured in appropriate culture
media. The probe PHS1 (10 μM) were incubated with cells for 30 min.
The treated cells were washed with PBS three times. Then, the cells
2
.5. Cell Culture and Confocal Fluorescence Imaging for Endogenous H
O
2 2
were co-incubated with H
2
O
2
(200 μM) and H S (200 μM) 30 min, the
2
and H S in Living Cells
2
culture media were removed and washed with PBS-buffer. Leica TCS
SP5 X AOBS Confocal Fluorescence Microscope (Germany) with a 63×
oil-immersion objective lens was used for fluorescence imaging. Cell
images were collected from the blue channel (450–480 nm) and green
channel (540–580 nm) at the excitation wavelength 400 nm.
The HT-29 cells and HeLa cells were received from the Food
Industry Research and Development Institute (Taiwan). The HT-29 and
HeLa cell line were grown in McCoy's 5A and DMEM Medium with 10%
(
v/v) FBS (fetal bovine serum) and penicillin/streptomycin (100 μg mL-
1
) at 37 °C in a 5% CO
2
incubator respectively. The endogenous H S
2
deduction was optimized through the HT-29 cells, which are treated
with 10 μM of PHS1 in DMSO and incubated for 1.30 h at 37 °C. The
green fluorescence in cells increased significantly, indicating the gen-
2.8. Zebrafish Imaging
The zebrafish were used as per the instructions of the National
eration of endogenous H
2
S within the cells. Also, for endogenous H
O
2 2
−1
Institute of Health Taiwan, and acceptance of the Institutional Animal
Care and Use Committee (IACUC) of National Chiao Tung University.
The treatment of the zebrafish was done according to the direction for
Animal Care and Use Committee of National Chiao Tung University.
The zebrafish were maintained at an optimal breeding condition of
around 28 °C. Under a 14 h light/10 h dark cycle, two male and female
zebra fish were maintained in one tank at 28 °C for mating. The next
morning, the laid eggs were triggered by light stimulation. After im-
mediate fertilization of most of the eggs, the zebrafish were cultured in
embryo medium (5 mL) with added 1-phenyl-2-thiourea (PTU) in 6-
well plates for 24 h at ambient temperature. Immediately, the an-
esthetized (50 mg L-1 tricaine) zebrafish embryos were incubated with
deduction in the HeLa cells, we incubated with PMA (25 ng mL ) for
h and then PHS1 (10 μM) for 30 min at 37 °C. The culture medium
was removed, and the treated cells were washed with 0.1 M PBS
2
(
2 mL × 3) before observation. The fluorescence images of cells were
collected with a Leica TCS SP5 X AOBS Confocal Fluorescence
Microscope (Germany) fitted with a 63 oil-immersion objective lens.
The emissions bands were varied from 460 nm and 550 nm ± 10 nm
respectively.
2
.6. Confocal Fluorescence Imaging for Exogenous H
2
O in HeLa Cells
2
Cells were cultured in Dulbecco's modified Eagle's medium (DMEM)
H
2
S and H
2
O separately in E3 media for 30 min at ambient tempera-
2
supplemented with 10% fetal bovine serum (FBS) at 37 °C under 5%
CO atmosphere. After 24 h, the cells were washed with PBS and then
incubated with 10 μM of PHS1 in 2 mL DMEM for 30 min. Then, H
150 μM) was added in culture media and incubated for 30 min. The
culture medium was replaced, and the treated cells were washed with
.1 M PBS (2 mL × 3). The fluorescence images of cells were acquired
ture. Several PBS washes removed the excess H
2
S and H
2
O and then
2
2
zebrafish were additionally incubated with PHS1 (50 μM) for 30 min.
Subsequent washes with PBS were done to remove the surplus probe
and then the imaging of zebrafish was done using a Leica TCS SP5 X
AOBS Confocal Fluorescence Microscope.
O
2 2
(
0
101

N. Velusamy et al.
Journal of Photochemistry & Photobiology, B: Biology 191 (2019) 99–106
Fig. 1. (a) Fluorescence spectra of PHS1 (10 μM)
with H
2
O (0–250 μM). Inset: Average fluorescence
2
intensity (λem = 460 nm) changes with H
Fluorescence response of PHS1 (10 μM) with H
200 μM) at time (0–70 min). c) Fluorescence re-
2
O . b)
2
O
2 2
(
sponse of PHS1 (10 μM) towards 200 μM a: HOCl, b:
Na
Na
Na
2
2
2
S. c: NaNO
g: FeSO
, l: GSH (1 mM), m: Ascorbic Acid (1 mM), n:
2
,
d: Tyrosinase, e: Zn(OAc)
2
, f:
S
2
O
3
,
4
,
h:NO i:Trypsin j: Pepsin k:
CO
3
Cysteine (1 mM), o: Folic Acid (1 mM), p: Histidine
(
1 mM), q: Lysine (1 mM), r: NaOCl s: K
. d) Average fluorescence intensity changes of
PHS1 (10 μM) at λex = 390 nm in presence and ab-
sence of H , (200 μM) at various pH (4–9.5). All
the experiments were carried out in PBS buffer
2
S and t:
5
H O
2 2
O
2 2
(
pH = 7.4; 1% DMSO) at 37 °C with excitation at
3
90 nm and slit width set at 5 nm.
Scheme 2. Reaction mechanism of PHS1 with H
2
O
2
, H
2
S and H
2
O
2
/H S.
2
3
. Results and Discussions
H
2
O
2
and H
2
S respectively. Further, the overlap of the emission band of
coumarin moiety with the absorption of naphthalimide (Fig. S3) has
3.1. Synthesis and Photophysical Properties of PHS1
suggested that PHS1 can provide information on temporal crosstalk
between H
2
O
2
and H S in living cells through FRET-based fluorescence
2
We synthesized probe PHS1 as outlined in Scheme 1. All the in-
imaging. Based on the above design strategy, 2we believe that PHS1
1
13
termediates and probe PHS1 were characterized by H NMR, C and
MS analysis (ESI†). The detail of the synthetic procedure and the
spectroscopic information is available in the experimental section. In
this design strategy, we have chosen coumarin and naphthalimide as
the fluorophore because their distinct absorption in 390 and 460 nm
could provide information on H , H S, and H /H S.
2
O
2
2
2
O
2
2
We then incubated PHS1 with H
2
2
O (200.0 μM) for 1 h to know the
UV-abs. Changes of PHS1. As we noticed in the Fig. S4, UV-abs. at
390 nm has 35-fold enhanced. Next, we titrated PHS1 with variable
concentrations of H
2
O
2
(0–200.0 μM), as depicted in Fig. 1a, the
(
Fig. S1) can separate analytes H
2
O
2
and H
2
S respectively. Moreover,
fluorescence intensity at λem 460 nm is continuously increased with the
emission maxima for coumarin and naphthalimide fluorophore also
have a distinctive range of 450 nm and 550 nm (Fig. S2) respectively;
thereby, the two mentioned analytes can be detected selectively
without any interferences. By introducing two fluorophores in PHS1, it
is possible to acquire dual-colour images in living cells for the analytes
increasing levels of H
2
O
2
O
. From the regression equation, obtained de-
−5
tection limit towards H
2
2
was 12.1 × 10 μM (Fig. S5). The result of
time-dependent (Fig. 1b) titration of PHS1 with H
2
O indicated that 7-
2
pinacolboronyl- coumarin moiety in PHS1 was fully converted to 7-
hydroxy coumarin derivative FLH2O2 within 60 min [8] (Scheme 2, Fig.
102

N. Velusamy et al.
Journal of Photochemistry & Photobiology, B: Biology 191 (2019) 99–106
Fig. 2. a) Concentration dependent study of PHS1
(
10 μM) with Na S (0–250 μM). Inset: Concentration
2
vs average fluorescence intensity (λem = 550). b)
Fluorescence response of PHS1 (10 μM) with Na
200 μM) at time (0–70 min). c) Fluorescence re-
sponse of PHS1 (10 μM) towards 200 μM a: HOCl, b:
2
S
(
H
2
O
2
,
c: NaNO
, g: FeSO
, l: GSH (1 mM), m: Ascorbic Acid (1 mM), n:
2
,
d: Tyrosinase, e: Zn(OAc)
2
, f:
Na
2
2
S
2
O
3
3
4
, h:NO i: Trypsin j: Pepsin k:
Na
CO
Cysteine (1 mM), o: ALP (1 mM), p: Histidine
(
1 mM), q: Lysine (1 mM), r: NaOCl s: K
Na S. d) Average fluorescence intensity changes of
PHS1 (10 μM) in presence and absence of Na
200 μM) at various pH (4–9.5). The spectra were
recorded in PBS buffer (pH = 7.4; 1% DMSO) at
2
S and t:
5
2
2
S
(
3
7 °C and excitation was effected at 450 nm; both
excitation and emission slit widths set at 5 nm.
Fig. 3. a) Fluorescence intensity changes of PHS1
(
10 μM) in the absence and presence of both H
2 2
O
and H
sponse of PHS1 (10 μM) towards 200 μM a: HOCl, b:
, c: NaNO , d: tyrosinase, e: K , f: Na , g:
, h: NO i: pepsin j: CaCl k: NaOCl, l: GSH
1 mM), m: ascorbic Acid (1 mM), n: cysteine
2
S (200 μM) at pH (7.4). b) Fluorescence re-
H
2
O
2
4
2
2
S
5
2
S
O
2 3
FeSO
2
(
(
1 mM), o: ALP (1 mM), p:
H
2
O
2
and
H S.
2
Experiment was executed with 1% DMSO at 37 °C
with excitation at λex = 400 nm.
S6). Also, this process is free from interference from biologically re-
levant analytes and pH, as fluorescence intensity of PHS1 has remained
invariable in the presence of various analytes including pH (Fig. 1c-d).
H
2
S/H
2
O
2
incubated PHS1 solution while excitation was effected at
400 nm. The small peak at 450 nm indicated that the FRET mechanism
is partially perturbed. This FRET-based fluorescence signal was ob-
On the other hand, upon incubation of PHS1 with H
2
S (Na
2
S), the
tained because of the formation of FL-1 (Scheme 2); it was confirmed
1
UV-abs. at λabs 460 nm (Fig. S4) has increased around 32-fold and the
by HRMS analysis and H NMR (Fig. S9–10) of H
2
S/H
2
O pretreated
2
fluorescence intensity at 550 nm has increased with rising levels of H
2
S
PHS1. Notably, this crosstalk event is free from interference from other
(
Fig. 2a). It is obviously due to the formation of 8-aminonapthalimide
analytes (Fig. 3b). All the experimental results led us to conclude that
derivative FLH2S (Scheme 2, Fig. S7). PHS1 has also shown time-de-
the probe PHS1 could detect H
2
S, H
2
O
2
, and H
2
S/H
2
O by providing
2
pendent fluorescence enhancement and reaches saturation within
multicolor fluorescence images in live cells.
6
0 min in the presence of 200 μM of H
2
S (Na S).
2
Notably, this H S sensing event is free from interference from other
analytes, including pH (Fig. 2c-d). Indeed, the reactivity of PHS1 to-
wards H S is pH-dependent and showed strong fluorescence intensity
2
3.2. Cell Viability and Tracking of H S, H , and H
2
2
O
2
2
S/H
2
O in Live Cells
2
2
The probe PHS1 had shown negligible cytotoxicity at very high
within pH 6–8.5 range (Fig. 2d). By using the regression equation, the
S was found to be 52.3 × 10−5 μM
concentrations (≥ 50 μM) against HT29 cells, but it is nontoxic within
the range of levels used for cell imaging (≤ 10 μM) (Fig. S11). Such
non-toxic nature of PHS1 encourages using it in vitro tracking of en-
detection limit of PHS1 towards H
2
(
Fig. S8).
We then established the crosstalk between H
2
S and H
2
O through
2
dogenous analytes, such as H
2
S, H
2
O
2 2
, and H S/H
2
O respectively.
2
FRET-based fluorescence study. As depicted in Fig. 3a, a fluorescence
signal at 550 nm with a small shoulder peak at 450 nm is observed in
Next, we had proceeded towards labeling of colon cancer cells (HT29)
based on endogenous H S. We noticed in Fig. 4e, the cells were green
2
103

N. Velusamy et al.
Journal of Photochemistry & Photobiology, B: Biology 191 (2019) 99–106
Fig. 4. Confocal fluorescence images of HT-29. (Left)
Bright field, (Middle) Fluorescence and (Right)
merged image. The cells were without PHS1 (a-c)
and with PHS1 (10 μM) were incubated with for
1
.30 h (d-f). Images were collected from the emission
wavelength green channel (540–580 nm) at the ex-
citation wavelength 460 nm. Scale bar = 25 μm. (For
interpretation of the references to colour in this
figure legend, the reader is referred to the web ver-
sion of this article.)
Fig. 5. Detection of PMA-induced H
in HeLa cells. (a-c) cells were incubated with PHS1
10 μM) for 30 min. (d-f) Subsequent treatment of
the cells with the stimulant PMA (25 ng mL ) for
2
O
2
production
(
−
1
2
h. Images were collected from the emission wave-
length blue channel (450–480 nm) at the excitation
wavelength 400 nm. Scale bar = 50 μm. (For inter-
pretation of the references to colour in this figure
legend, the reader is referred to the web version of
this article.)
Fig. 6. Confocal fluorescence images of HeLa cells. (a-c) cells were incubated with PHS1 (10 μM) for 30 min. (d-f) Subsequent treatment of the cells with H
150 μM) for 30 min. Images were collected from the emission wavelength blue channel (450–480 nm). Scale bar = 25 μm.
O
2 2
(
104

N. Velusamy et al.
Journal of Photochemistry & Photobiology, B: Biology 191 (2019) 99–106
Fig. 7. Confocal fluorescence images of HeLa cells.
The probe PHS1 (10 μM) were incubated for 30 min
and then treated (a-d) both H
2
O
2
(200 μM) and H S
2
(
200 μM). Images were collected from the emission
wavelength blue channel (450–480 nm) green
channel (540–580 nm) at the excitation wavelength
4
00 nm. Scale bar = 50 μm. (For interpretation of
the references to colour in this figure legend, the
reader is referred to the web version of this article.)
Fig. 8. Confocal microscopy images of 5-day-old
zebrafish (Left) Bright field; (Middle) Fluorescence;
and (Right) merged image. (a-c) the zebrafish were
without PHS1. (d-f) the zebrafish were incubated
with PHS1 (50 μM) for 30 min and then with H S
2
(
150 μM) for 30 min. (g-i) the zebrafish were in-
cubated with PHS1 (50 μM) for 30 min and then with
(150 μM) for 30 min. Images were collected at
the blue (450–480 nm) and green channels
H
O
2 2
(
540–580 nm) with 400 and 450 nm excitation, re-
spectively. Scale bar = 750 μm. (For interpretation
of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
fluorescence labeled upon incubation with probe PHS1. This green
cells by acquiring FRET based images in the green fluorescence channel.
As shown in Fig. 7, there was virtually no fluorescence in the blue
fluorescence image is because of the conversion of N
3
- to H
2
N- in the
naphthalimide moiety of PHS1 in the presence of endogenous H
2
S [21].
channel region upon exciting at λex 400 nm in H
2
O
2
/H S pretreated
2
On the other hand, there were no fluorescence signals either in blue or
HeLa cells; instead, in the green channel marked fluorescence was no-
green channel upon excitation at 400 nm/450 nm in PHS1 incubated
ticed (Fig. 7c). These findings are well consistent with our designed
HeLa cells; evidently, it is due to insufficient endogenous H
2
O
2
/H
2
S
strategy for tracking of H
2
O
2
, H
2
S and H
2
O
2
/H S formation in cancer
2
formation.
cells through multicolor fluorescence imaging. The dual analytes (H
O
2 2
and H S) sensing in cancer cells could be a unique strategy for diag-
2
nosing cancer cells which may pave the way to develop new ther-
apeutics in cancer treatment.
3.3. Fluorescence Imaging
We were then interested in the tracking of stimulator-induced en-
dogenous H formation in HeLa cells. Thus, PHS1 pretreated HeLa
cells were further incubated with phorbol 12-myristate 13-acetate
PMA) to induce endogenous H formation; as a result, bright
fluorescence images were obtained in the blue channel (Fig. 5e). It is
apparently due to the formation of endogenous H , as noticed in the
previous study [47]. Similarly, when PHS1 was treated with exogenous
(150 μM) for 30 min, it provided blue fluorescence images.
Fig. 6). It indicates that the probe PHS1 can detect both exogenous and
endogenous H in the live cells.
We then proceed to check the presence of both H /H S in cancer
2
O
2
3.4. Tracking of Exogenous H
2
S, and H
2
O in Live-Specimen
2
(
O
2 2
Finally, we examined the in vivo H
2
O
2
and H S formation in the
2
Zebrafish life specimen. As shown in Fig. 8e-f, the probe PHS1 treated
O
2 2
Zebrafish is labeled in green fluorescence channel in the presence of
H
2
S. On the other hand, Zebrafish coincubated with PHS1/H
2
2
O are
H
O
2 2
blue fluorescence labeled (Fig. 8h-i). From this result, we can conclude
(
that probe PHS1 can validate both H
2
O
2
and H S formation in the live
2
O
2 2
specimen.
2
O
2
2
105

N. Velusamy et al.
Journal of Photochemistry & Photobiology, B: Biology 191 (2019) 99–106
4
. Summary and Conclusions
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