Superb-selective chemodosimetric signaling of sulfide in the absence and in the presence of CT-DNA and imaging in living cells by a plant alkaloid berberine analogue

Article abstract of DOI:10.1039/c8nj06120c

The present manuscript reports a lucrative design of a colorimetric and ratiometric chemodosimeter, 9-O-(2,4-dinitrobenzenesulfonyl)berberrubine (BER-S), with excellent water solubility for the superb selective detection of sulfides through a color alteration from yellow to red with a good limit of detection (LOD) of 56 nM in CP buffer solution (10 mM, pH 7.2). Interestingly, this work also includes a smart "turn-on" emission probe (BER-S/DNA complex) showing good linearity with an excellent LOD of 46 nM for recognizing sulfide anions. The probes, BER-S, and BER-S/DNA complex, displayed no interfering effect by other analytes or sulfur-containing inorganic compounds, like thiols. Characterization was carried out using IR, HRMS, and DFT for the BER-S probe, and time-resolved fluorescence lifetime measurement and fluorescence titration for the BER-S/DNA complex probe for elucidating their sensing mechanism. The detection of S^2- in waste, tap, and drinking water by BER-S indicated its potential application in real sample analysis, while concentration variant cell imaging experiments (naked-eye red fluorescence) verified its cell-membrane permeability and capability for S^2- imaging in living cells. This reaction-based sensing strategy in the presence of DNA may provide a potential platform for the design of a fluorescent chemodosimeter for extensive anion targets.

Full text of DOI:10.1039/c8nj06120c

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Superb-selective chemodosimetric signaling of
sulfide in the absence and in the presence of
CT-DNA and imaging in living cells by a plant
alkaloid berberine analogue†
Cite this:DOI: 10.1039/c8nj06120c
a
a
a
a
a
Gopal Chandra Jana, Munira Khatun, Sk Nayim, Somnath Das, Anukul Maji,
a
a
b
b
Maidul Beg, Anirudha Patra, Paromita Bhattacharjee, Kakali Bhadra and
a
Maidul Hossain
*
The present manuscript reports a lucrative design of a colorimetric and ratiometric chemodosimeter,
-O-(2,4-dinitrobenzenesulfonyl)berberrubine (BER-S), with excellent water solubility for the superb
9
selective detection of sulfides through a color alteration from yellow to red with a good limit of
detection (LOD) of 56 nM in CP buffer solution (10 mM, pH 7.2). Interestingly, this work also includes a
smart ‘‘turn-on’’ emission probe (BER-S/DNA complex) showing good linearity with an excellent LOD of
4
6 nM for recognizing sulfide anions. The probes, BER-S, and BER-S/DNA complex, displayed no
interfering effect by other analytes or sulfur-containing inorganic compounds, like thiols. Characterization
was carried out using IR, HRMS, and DFT for the BER-S probe, and time-resolved fluorescence lifetime
measurement and fluorescence titration for the BER-S/DNA complex probe for elucidating their sensing
2
ꢀ
Received 3rd December 2018,
Accepted 3rd January 2019
mechanism. The detection of S
in waste, tap, and drinking water by BER-S indicated its potential
application in real sample analysis, while concentration variant cell imaging experiments (naked-eye red
2ꢀ
fluorescence) verified its cell-membrane permeability and capability for S imaging in living cells. This
reaction-based sensing strategy in the presence of DNA may provide a potential platform for the design of
a fluorescent chemodosimeter for extensive anion targets.
DOI: 10.1039/c8nj06120c
rsc.li/njc
ecosystems and human beings, and their concentration is an
essential aspect of the environmental index. Sulfides can cause
1
. Introduction
3
The design and development of efficient sensor systems to coma and respiratory paralysis, and even infuriate mucous
2
,4
ꢀ
detect various chemically and biologically significant species membranes. The protonated form of sulfides, i.e., HS or
has attracted considerable interest from researchers. Recently,
1
2 2
H S, are more toxic than sulfides. H S at low concentration can
the potential of a high recognition and sensitive anion detec- produce individual distress; however, higher concentrations
tion platform has drawn considerable attention for their vital cause the loss of perception, eternal brain spoil, or even death
1
b
5
roles in environmental, industrial, and biological processes.
through asphyxiation. Furthermore, sulfides have emerged as
Being one of the most environmentally and biologically signifi- a novel vital mediator in the cardiovascular organism, the
6
cant anions, sulfides are frequently used in various fields, nervous system, and in various biological signaling functions.
for example, dyes and cosmetic manufacturing, for conversion Therefore, it is challenging to develop a water-soluble, rapid,
2
into sulfuric acid, and in the production of wood pulp. As a reliable, and sensitive probe for the detection of sulfide anions
2
ꢀ
result, sulfide (S ) anions can be widely circulated in water via in aqueous medium and biological systems.
industrial processes and by the reduction of sulfate by anaerobic
bacteria. Besides, sulfides have a harmful effect on aquatic for S
To date, many detection techniques have been developed
2
2ꢀ
7
anions; for example, electrochemical methods, ion
8
9
chromatography, titration, inductively coupled plasma
1
0
11
atomic emission spectroscopy, spectrophotometry, hydride
generation atomic fluorescence spectrometry, fluorometric,
and chemiluminescence. While they offer high accuracy, these
methods need complicated instrumentation, expert personnel,
and time-consuming sample preparation, which make them
inappropriate for real applications. The fluorescent sensing
a
12
13–17
Department of Chemistry and Chemical Technology, Vidyasagar University,
Midnapore-721102, West Bengal, India. E-mail: hossainm@mail.vidyasagar.ac.in;
Tel: +91-9432976277
18
b
Department of Zoology, University of Kalyani, Kalyani-741235, West Bengal, India
Electronic supplementary information (ESI) available. See DOI: 10.1039/
†
c8nj06120c
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method has received great attention among these reported recog-
nition methods due to its high sensitivity and simple operation.
Till now, many fluorescent probes have been successfully
1
9–27
developed to detect sulfide anions.
However, most of these
probes suffer from poor water solubility, i.e., a mixture of water
and the organic solvent is required for their better sensitivity,
which limits their application in environmental and bio-related
2
8–30
fields.
As a result, a lot of improvements are needed for
sulfide anion detecting probes for potential environmental and
biological applications, such as excellent water solubility and
3
1–35
appliance in biological systems.
The sulfide anion is an effective nucleophile and reacts
readily with sulfonate esters or sulfonate ether compounds,
and this fact tunes the idea for the design of a novel turn-on
2
ꢀ
36–41
fluorescent chemodosimeter for S anions.
In the recent
literature, a few probes have been designed by splitting the Scheme 1 Schematic of the proposed sensing mechanism in the
absence of DNA and in the presence of DNA by BER-S.
dinitrophenyl ether or dinitrobenzenesulfonyl group to bring
4
2–44
back their original fluorescence.
Thus, it was expected that
incorporating the strong electron-withdrawing group dinitro-
benzenesulfonate ester into a fluorophore could appreciably
reduce the fluorescence, and then by cleaving it, a significant
selective detection of sulfide anions in CP buffer solution
10 mM, pH = 7.2) (Scheme 1). Additionally, concentration
variant confocal microscopic imaging revealed that the BER-S
was cell-permeable and sensed S in the living cell. Thus, it
heralds a new platform for the detection of sulfide anions by a
DNA–alkaloid template and has unique biological value due to
its ability to permeate the cell membrane and detect sulfide
anions in a living cell.
(
4
5–48
amplification in fluorescence intensity could be observed.
2
ꢀ
More recently, a fluorescent DNA–gold/silver nano-clusters
template was developed that allowed the discerning detection
4
9
of sulfide anions, having a detection limit of 0.83 nM, while
2
ꢀ
another fluorescent sensor for the rapid detection of S using
5
0
dsDNA–CuNPs as fluorescent probes was also reported. We
recently developed a new sensing approach in the presence
of DNA for hydrazine sensing with superb selectivity using a
2
. Experimental section
5
1
DNA–BER-BZ complex as a novel fluorescence probe. In this
system, hydrazine cleaved the ester bond of the probe to
form an hydrazinolysed compound that also interacts with
the DNA, and this interaction concomitantly causes a great rise
in fluorescence intensity. Although the use of such a DNA–
alkaloid complex in sensing has been barely studied, such
potential emission probes have drawn immense attention for
their potential biochemical impact. Thus, application of the
DNA–alkaloid complex is of significant interest for sensing
studies, particularly for application in highly selective and
sensitive sulfide detection.
2
.1. Materials/chemicals and reagents
Berberine chloride (BC), sodium sulfide (Na
thymus (CT) DNA (type XI, 42% guanine–cytosine content) were
purchased from Sigma-Aldrich Corporation (St. Louis, MO,
2
Sꢁ10H
2
O), and calf
2
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
2ꢀ
ꢀ
USA). Anionic salts (S , Cl , Br , I , F , Ac , NO , NO3
,
,
2
2ꢀ
3
ꢀ
ꢀ
ꢀ
ꢀ
2ꢀ
2ꢀ
PO4 , CN , SCN , HSO , SO , SO , S O , S O
3
4
3
2
3
2
8
HPO42 , H PO , ClO , IO , S O
ꢀ
ꢀ
ꢀ
ꢀ
2ꢀ, BO33ꢀ
2+
, B O , N3 ),
4 7
2ꢀ
2
4
4
4
2
8
+
+
2+
2+
2+
3+
2+
2+
cationic salts (Na , K , Ca , Mg , Pb , Cu , Fe , Hg , Zn ,
Cd , Co ), and life elements, such as ATP, GTP, ClO , H O ,
2 2
tyrosine, tryptophan, lysozyme, including biothiols (glutathione,
cysteine, homocysteine, methionine, cysteamine, and thiobarbi-
turic acid), and other solvents were obtained from Merck Pvt. Ltd
2
+
2+
ꢀ
Inspired by the above perspective, we report herein a turn-on
2
ꢀ
fluorescence sensor for S ions using the BER-S/DNA complex.
(Singapore) and were used directly without further purification.
Accordingly, we synthesized a new 9-O-berberine derivative
The concentrations of BER-S and CT-DNA were determined by
(
2
BER-S, Scheme 1) by substituting the electron-withdrawing
,4-dinitrosulfonyl group at the 9-O position in berberine.
ꢀ1
ꢀ1
molar extinction coefficient (e) values of 13 200 M
at 260 nm and 19000 M cm at 342 nm, respectively. The
CT-DNA was dissolved in CP buffer solution (10 mM [Na] at
cm
ꢀ
1
ꢀ1
Owing to the presence of a sulfonate ester linkage at the 9
position, the probe acted as an excellent colorimetric chemo-
dosimeter for sulfide anions in CP buffer medium (Scheme 1).
The analogue BER-S could interact with DNA, and this binding
phenomenon was investigated by various spectroscopic meth-
ods, such as UV-vis, fluorescence, fluorescence quenching, and
viscosity measurements, which revealed that the analogue has a
+
pH 7.2) and the purity of DNA was confirmed by monitoring the
ratio of absorbance at 260/280 nm (A260/A280).
2.2. Instrumentation
1
13
1
H (500 MHz) and C{ H} (125 MHz) NMR spectra were
higher binding efficacy with CT-DNA than berberine. More recorded on a Bruker’s AVANCE-III 500 MHz spectrometer in
interestingly, the probe in a DNA-binding situation (maximum DMSO-d solvent using TMS as an internal reference at 25 1C.
6
saturation), i.e., the BER-S/DNA complex, can act as a potential Mass spectra of compound BER-S and mass spectral data for
turn-on chemodosimetric fluorescence probe for the highly the sensing study were measured using a BrukermicroTOF QII
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high-resolution mass spectrometer coupled to a Waters Acquity at 500 mM in Millipore water. The solution of BER-S was then
UPLC system. FT-IR spectra were measured with a PerkinElmer diluted to 1 mM with CP buffer (10 mM, pH = 7.2). The titration
Spectrum, two FT-IR spectrophotometers equipped with a zinc experiments were carried out using 1 ml of BER-S (10 mM) stock
selenide (ZnSe) attenuated total reflectance (ATR) accessory, solution of the probe, with BER-S in a quartz cell of 1 cm path
LiTaO detector, and a KBr beam splitter at ambient temperature length, and various analytes were gradually added by micro-
3
(PerkinElmer, Inc., USA). UV-vis spectra were obtained from a pipette to record the spectrum. Spectrophotometric and spectro-
Shimadzu UV-1601 spectrophotometer (Shimadzu Corporation, fluorimetric titrations of BER-S/DNA complex were performed
Kyoto, Japan). All the spectra were collected at 25 1C in quartz taking solution (10 mM probe, 150 mM CT-DNA) in a quartz cell
cuvettes of 1 cm path length. All the fluorescence spectra were containing CP buffer (pH = 7. 2) with analyte solution added to
measured on a PerkinElmer LS-55 (USA) spectrofluorimeter in record the spectra. For the fluorescence experiments, the excita-
fluorescence-free quartz cells of 1 cm path length. The widths of tion wavelength was fixed at 360 nm, and the emission was
the excitation slit and emission slit were set to 2.5 nm and 5 nm, recorded from 400 to 700 nm.
respectively.
2.6. Preparation of stock BER-S solution for the binding study
The fluorescence lifetime data were collected using a
DeltaFlext Moduler Fluorescence Lifetime system (Horiba Stock solutions of BER-S were prepared in CP buffer solution
Scientific, UK). The decay data were measured by the time- and kept in the dark to avoid light-assisted photophysical
correlated single photon counting (TCSPC) procedure using CP changes. In this study, BER-S obeyed Beers’ law in the experi-
buffer of pH 7.2 at an excitation wavelength of 372 nm from a mental concentration range. The concentration of the BER-S
laser LED source.
solution was measured by the molar extinction coefficient (e)
Density functional theory (DFT) and calculations via value of 19 000 at 342 nm from the absorbance study.
CAM-B3LYP/6-31g(d,p) level using the Gaussian 09 program were
2
.7. Procedure for UV-vis and fluorescence spectral
performed. A rotational viscometer (Brookfield, Middleboro, MA
2346, USA) armed with a 1 ml LCP spindle was used for the
viscosity experiments, operating at 40 rpm at 25 1C.
measurements for the binding study
0
The absorption titration experiments were carried out with
gradually increasing the concentration of CT-DNA to a fixed
concentration of BER-S. In general, to a solution of BER-S
2.3. Synthesis of berberrubine (2)
Commercially available berberine (1, 1 g) chloride was heated at (10 mM) in 10 mM CP buffer were added aliquots of CT DNA
90 1C in a vacuum oven under reduced pressure of 20–30 mm in 10 mM CP buffer up to saturation, according to a previously
1
5
2
Hg for 15 min to get berberrubine (2, 885 mg) in about a 90% reported protocol. The emission titration was performed as
yield according to previously reported work.
5
1
dictated in the absorbance titration taking BER-S (10 mM) in
0 mM CP buffer solution.
1
2
.4. Synthesis of 9-O-(2,4-
dinitrobenzenesulfonyl)berberrubine (3)
First 2,4-dinitrobenzenesulfonyl chloride was added to the The fluorescence quenching experiment was performed with
solution of 2 in CH CN, and then the reaction mixture was
[Fe(CN) ], an anionic quencher as per a previously reported
2.8. Fluorescence quenching experiments
K
3
4
6
3
5
permitted to stir for 30 min at room temperature. The crude protocol. The K [Fe(CN) ] and KCl solutions were mixed in
4 6
product was obtained by evaporating the solvent under vacuum changed ratios to give a fixed total ionic strength. According
and washed with diethyl ether. The product was purified to the Stern–Volmer equation, the data were plotted as the
through column chromatography on a silica gel column, eluted relative fluorescence intensity with varying concentration of the
with varying concentrations of the CHCl
3 3
–CH
OH solvent mix- ferrocyanide at a constant P/D (CT-DNA/BER-S molar ratio)
4
ꢀ
ture to get the desired product BER-S (3) as a yellow powder versus [Fe(CN)
6
] .
1
(
80–85% yield), H NMR (500 MHz, DMSO-d
6
, TMS, 25 1C)
2.9. Viscosity measurements
d = 3.22 (2H, t, J = 10), d = 3.83 (3H, s), d = 4.99 (2H, t, J = 7.5),
d = 6.18 (2H, s), d = 7.11 (1H, s), d = 7.18 (1H, s), d = 8.10 (1H, d, To further inspect the binding mode of the BER-S with DNA,
J = 10), d = 8.22 (1H, d, J = 10), d = 8.32 (1H, d, J = 10), d = 8.54 viscosity measurements were performed on CT-DNA with respect
(
1H, s), d = 8.72 (1H, d, J = 10), d = 9.16 (1H, s), d = 9.65 (1H, s). to varying concentrations of BER-S. The viscosities of a DNA
1
3
C NMR (125 MHz, DMSO-d
, TMS, 25 1C) d 29.83, 56.19, 59.24, solution (1 mM) were measured in the absence and presence of
6
1
1
1
01.26, 103.14, 110.80, 112.68, 115.12, 118.81, 121.51, 124.26, increasing concentrations of the BER-S (0.05–0.25 mM). The
28.59, 131.96, 135.37, 137.52, 140.22, 142.74, 145.18, 146.99, relation between the relative solution viscosity (Z/Z
) and DNA
0
1
54
48.23, 151.02, 153.45, 156.24, 157.42. The values of H, and length (L/L
) is given by the following equation:
0
1
3
C NMR are in ppm units. UV/Vis (H O): 406, 342, 259 nm.
2
1/3
L/L = (Z/Z )
0
0
HRLC-MS for C H N O S calcd, 552.07, found 552.0765.
2
5
18 3 10
where L and L denote the apparent molecular length in the
presence and absence of the compound, respectively. The
0
2
.5. Procedure for spectral measurement for sulfide detection
1
/3
The solutions of various analytes (1 mM) were prepared in obtained data are presented as (Z/Z
0
)
versus r, where Z is
is the
Millipore water. The stock solution of the BER-S was prepared the viscosity of DNA in the presence of BER-S, and Z
0
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viscosity of DNA alone in a buffer solution, and r represents the DMEM medium supplemented with 10% heat-inactivated FBS
ratio of [BER-S]/[DNA]. and 1% antibiotic–antimycotic solution, in a CO incubator at
2
4
3
3
7 1C with 5% CO . After culturing the cells (2 ꢃ 10 cells per
2
2.10. Analysis of the binding data
5 mm culture plate) for 24 h, the probe was added in various
The UV-visible and fluorometric titrations were carried out concentrations (5, 10, 15, 20, and 25 mM) and again after 48 h
in the range of 300–600 nm and 400–660 nm, respectively, in incubation. The percentage of viable cells was calculated as
CP buffer solution to calculate the apparent binding constant the IC50 (50% growth inhibition) values for the cell lines
ꢀ
1
from Scatchard plots of r/C versus r (where r is the number of by MTT (1 mg ml of the MTT) assay kit (Sigma), by the
f
moles of alkaloid analogue bound per mole of CT-DNA base following formula:
pairs binding). The Scatchard plots showed a negative slope
at higher r valued as observed in non-cooperative binding
IC50 = (T ꢀ T
0
) ꢃ 100/(C ꢀ T
0
)
isotherms and therefore the binding affinity was analyzed by
the following McGhee–von Hippel equation:
where T represents the optical density of the sample culture
5
5
plate after 48 h of probe treatment, T is the optical density at
time zero, and C is the optical density of the control.
0
r
¼
Kið1 ꢀ nrÞ½ð1 ꢀ nrÞ=f1 ꢀ ðn ꢀ 1Þrgꢂnꢀ1
Cf
2
.14. Fluorescence cell imaging
where K is the intrinsic binding constant to an isolated binding
i
We performed the concentration variant fluorescence cell ima-
ging experiment with the A375 (skin melanoma) cell line. After
site and n is the number of base pairs excluded by the binding of
a single alkaloid molecule. All the binding data were analyzed
through the origin software, from which we calculated the best-
4
culturing the cells (2 ꢃ 10 cells per 35 mm culture plate) for
2
4 h, the various concentration of BER-S (3, 6, and 10 mM) was
i
fit parameters K and n.
added in the three set of samples and after 30 min incubation,
the images were obtained using an Invitrogen FL Digital
Inverted Fluorescence Microscope (Invitrogen AMF 4300). Then
2.11. Fluorescence quantum yield in solution
The fluorescence quantum yields of both the reactant (BER-S)
and cleaved product (BER-OH) in the absence and presence of
DNA were calculated in HPLC grade water using optically same
solutions of quinine sulfate (F = 0.546 in 1 N H SO ) as the
2ꢀ
the cells were treated with three different concentration of S
for 30 min interval for each set of samples and cell images were
obtained using the same fluorescence microscope stated above.
In all the cases, the red channel (540–644 nm wavelength range)
was chosen to obtain the fluorescence images.
r
2
4
standard at an excitation wavelength of 365 nm. The quantum
5
6
yield was calculated using the following equation:
2
2
F
s
= F
r r s s r s r
(A F /A F )(Z /Z )
3
. Results and discussion
where A and A are the absorbance of the reference and sample
r s
3.1. Design and synthesis of BER-S
solutions, respectively, and F and F are the corresponding
integrated fluorescence intensities (areas) at the same excita- One of the most interesting features of the sulfide detector
r
s
tion wavelength, and Z is the refractive index of the solvents.
probe is that it contains a strong electron-withdrawing group,
as already discussed in the Introduction section. Accordingly,
we selected the 2,4-dinitrobenzenesulfonyl moiety for its well-
known reactivity toward sulfide or thiols and utilized this as a
sulfide detector probe in this article. Once the sulfonate is
hydrolyzed by sulfide, it restores the color in the absence of DNA,
while fluorescence can be observed in the presence of DNA.
As shown in Scheme 2, berberrubine (2) was synthesized by a
selective demethylation reaction of berberine (1), followed by
2.12. Calculation of the detection limit
The detection limits of the probe BER-S and BER-S/DNA
complex were calculated by the UV-vis titration and fluores-
cence titrations, respectively. To get the detection limit value,
BER-S (10 mM) was titrated with an increasing concentration of
2
ꢀ
S
(0–25 mM). Similarly, the BER-S/DNA complex was titrated
2ꢀ
with various concentration of S (0–20 mM). The respective
reaction with 2,4-dinitrobenzenesulfonylchloride in CH CN for
absorbance and fluorescence intensity of BER-S and BER-S/DNA
3
2ꢀ
about 30 min to produce the desired final product BER-S (3).
complex were measured 10 times without S anion, and the
standard deviation of a blank measurement was obtained. To
gain the slope, the absorbance ratio (A377/A342) and the emission
1
13
The product was well characterized by H, C NMR, and TOF-MS
Fig. S20–S22, ESI†).
(
2ꢀ
intensity at 530 nm were plotted as a concentration of S . Then,
the detection limit was calculated with the following equation:
detection limit = 3s/k, where s is the standard deviation of blank
2
ꢀ
3
.2. Spectral recognition study of S by BER-S
3.2.1. UV-vis spectroscopic studies. The UV-vis spectra of
measurement, and k is the slope between the absorbance ratios BER-S in CP buffer solution (10 mM, pH = 7.2) exhibited an
2
(
A
377/A342) and I530 versus [S ].
absorption maxima at 342 nm, which may due to p–p* electronic
transition. Upon the gradual addition of S , the absorption
maxima at 342 nm gradually decreases, while a new peak at
2ꢀ
2
.13. Cytotoxicity assay
The A375 (skin melanoma) cell line was obtained from the 377 nm gradually appears and additionally another new peak at
National Centre for Cell Science, Pune. Cells were grown in 490 nm arises with one distinct isosbestic point (358 nm),
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Scheme 2 Synthetic pathway for the synthesis of BER-S, 3.
suggesting that two species are in equilibrium (Fig. 1a).
3.2.3. Selectivity, sensitivity, and competitive ion study.
2
ꢀ
Such a type of remarkable change in the absorption spectra The superb selectivity of BER-S toward S was evaluated by
red-shift of 35 nm) allows the probe BER-S to be used for measuring the change in the UV-vis spectra in the absence
(
2
ꢀ
2ꢀ
the colorimetric detection of S anions with a color change and presence of S . Almost no change in absorption ratio
from yellow to red (Fig. S4, ESI†). This is attributed by the (A377/A342) was observed with the addition of environmentally
2
ꢀ
ꢀ
2ꢀ
2ꢀ
ꢀ
ꢀ
2ꢀ
3ꢀ
ꢀ
ꢀ
2ꢀ
ꢀ
2ꢀ
nucleophilic attack of S to the sulfonate ester linkage of the and biologically rich anions like Cl , Br , I , F , Ac , NO
2
,
,
ꢀ
3ꢀ
ꢀ
ꢀ
ꢀ
probe, removing the 2,4-dinitrobenzenesulfonyl group from NO3 , PO , CN , SCN , HSO , SO4 , SO3 , S O , S O
the BER-S to produce berberrubine (2), and this structural HPO4 , H PO , ClO4 , IO , S O , BO3 , B O , N3 , but
change tunes the above changes in the electronic spectra. upon the addition of S , a drastic change in absorbance
4
3
2
3
2ꢀ
2
ꢀ
8
2
ꢀ
ꢀ
ꢀ
ꢀ
2
4
4
2
8
4 7
2
ꢀ
Accordingly, we plotted the absorbance ratio (A377/A342) vs. spectra were observed (Fig. S3, ESI†). We also checked the
2
ꢀ
concentration of S , which exhibited a nonlinear S like curve, selectivity of our probe with the addition of cations, such as
2
+
2+
3+
2+
2+
2+
2+
2+
2+
+
+
and consequently a linear relationship was observed between Ca , Mg , Fe , Cu , Zn , Hg , Cd , Pb , Co , Na , K , and
2
ꢀ
ꢀ
A
2 2
377/A342 and the S concentration in the range 0.0–1.2 mM other life elements, like ATP, GTP, ClO , H O , tryptophan,
with a correlation coefficient value R = 0.99235 (Fig. 1b). Based trypsin, lysozyme, and accordingly no abrupt change in absorp-
on the IUPAC convention, the limit of detection value (3s/k) tion was observed (Fig. S5 and S6, ESI†). These results are
consistent with our expectation that the sulfonate ester group
concentration in drinking water. Thus, BER-S could be can selectively be cleaved by S only, not by other ions.
2
2ꢀ
was 56 nM, i.e., much lower than the WHO permitted S
5
7
2ꢀ
employed as a promising ratiometric chemodosimeter for the
Interestingly, we also examined whether the absorbance ratio
changed or not in the presence of biological thiols, i.e., glutathione
2
ꢀ
detection of S
.
3
.2.2. Fluorescence study. Upon the successive addition (GSH), cysteine (Cys), homocysteine (Hcy), methionine (Met),
2
ꢀ
of S to the probe (10 mM), no remarkable change in the cysteamine (Cyt), and thiobarbituric acid (TBA). However, no
fluorescence spectra was observed, only a slight increase in the remarkable change in absorption spectra was observed in the
emission intensity (Fig. S5, ESI†). Hence, BER-S could not serve presence of the thiol (Fig. S7, ESI†). Owing to the greater nucleo-
2
ꢀ
2ꢀ
as a fluorescence turn-on probe for the detection of S
.
philicity of S compared with GSH, Cys, Hcy, Met, Cyt, and TBA can
Fig. 1 UV-vis spectra of BER-S (10 mM) (a) with increasing concentration of sulfide (0–2.5 equiv.). (b) The plot of absorption ratios at 377 nm and 342 nm
of BER-S (10 mM) upon the addition of varied concentrations of sulfide (0–25 mM), where the absorption ratio of BER-S (10 mM) was linearly related to the
concentration of sulfide (0.0–1.2 mM) in CP buffer solution (inset).
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Fig. 2 (a) Absorbance ratio (A377/A342) of BER-S (10 mM) to various anions in CP buffer (10 mM, pH = 7.2). The blue bars represent the absorbance ratio of
the probe in the presence of anions (each of 25 mM). The orange bars signify the changes of the ratios that occur upon the consequent addition of 25 mM
2ꢀ
of S to the above solution, (b) time-dependent absorbance ratio (A377/A342) of BER-S (10 mM) with sulfide anions (25 mM) in CP buffer solution.
2
ꢀ
attack selectively sulfonate linkages. Thus the above result indicates how the binding situation facilitates the detection of S were
2
ꢀ
that BER-S could specifically detect S among the other anions.
Additionally, we also performed an ambitious experiment where
also investigated.
3.3.1. Absorption and emission spectral studies. The binding
2ꢀ
the absorbance ratio (A377/A342) after the addition of S in the of BER-S with CT-DNA was mainly investigated by an absorption
presence other ions in BER-S solution was compared to that of the spectroscopy titration methodology. In absorbance titration,
2ꢀ
absorbance ratio (A377/A342) in the presence of S anions only significant bathochromic (342 nm to 347 nm) and hypochromic
2
ꢀ
(
Fig. 2a). Therefore, the colorimetric detection of S by BER-S shifts (30.85%) were observed with three isosbestic points
could withstand any interfering effects by other competitive anions. clearly visible (Fig. S1a, ESI†), which revealed a strong inter-
.2.4. Kinetics study. We also performed a kinetics study of molecular association. We also investigated the DNA-binding
3
2ꢀ
BER-S (10 mM) upon the addition of S to scrutinize the time affinity of BER-S via emission spectroscopy. Accordingly, upon
required for nucleophilic attack by S to sulfonate linkages in excitation at 360 nm, the emission maxima shifted surprisingly
CP buffer solution (pH = 7.2). In this experiment, a stock solution from 490 nm to 530 nm, and the fluorescence intensity
2ꢀ
2ꢀ
containing BER-S and S (1: 2.5 equiv.) was shaken well before increased remarkably upon binding with DNA up to saturation
the spectral measurement and accordingly the absorption ratio (Fig. S1b, ESI†). Such a huge shift in the emission maxima of
(A377/A342) at 120 s intervals was measured. The result showed around 40 nm with enhanced intensity indicated a strong
that the absorption ratio gradually increased and attained the binding efficacy. The intrinsic binding affinity (K ) and num-
i
highest value at 24 min, and after that no considerable increase bers of base pairs excluded by the binding of single alkaloid
in absorption ratio was observed (Fig. 2b). Therefore, the probe molecules (n) were obtained by analysis using McGhee–von
2
ꢀ
could detect S after 24 min of its addition.
Hippel’s non-cooperative model of binding (Table S2, ESI†).
5
ꢀ1
3
.2.5. The pH stability. In consideration of whether our probe The binding constant K was 3.6 ꢃ 10 M , i.e., greater than
i
58
could also be useful with environmental and biological systems, we that of the parent berberine. Details of the CT-DNA binding
further examined the influence of pH toward the colorimetric studies are incorporated in the ESI.†
2ꢀ
detection of S by BER-S. We found that the absorption ratio
377/A342) of BER-S (10 mM) was more or less same for a wide range
of pH (4–10), and consequently in the presence of S , the
absorption ratio (A377/A342) was nearly the same in the pH range
3
.4. Elucidation of the mode of binding of the analogue BER-S
.4.1. By fluorescence quenching and viscosity measure-
ment methods. Berberine is known to be a DNA intercalated
(A
2ꢀ
3
5
9
4
–10 (Fig. S8, ESI†). The result indicates that the BER-S could be
alkaloid. We are able to understand the mode of binding of
the analogue through the fluorescence quenching method.
In the fluorescence quenching method, an anionic quencher,
e.g., ferrocyanide ion, is used to distinguish between intercala-
2ꢀ
applied to a wide range of pHs for the selective detection of S
.
Thus, we used pH 7.2 (near neutral pH) in the whole experimental
2ꢀ
procedures for the detection of S , as this is very necessary for
proving the environmental and biological applicability of the probe.
5
8
tion and the groove binding modes of a berberine analogue.
Further strong evidence about the mode of binding has come
from viscosity measurements. In intercalation binding, the
DNA helix lengthens and consequently the DNA viscosity
3
.3. CT-DNA binding affinities studies by spectroscopic
analysis
As our synthesized probe BER-S could detect sulfide anions increases, whereas in the case of partial or non-classical inter-
in DNA-binding situations, the DNA-binding aspects and calation, the molecule can bend the DNA helix, shrinking its
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2
ꢀ
effective length with an attendant decreases in its viscosity, and linear relationship was noticed between I530 and S concen-
then the groove binding molecule does not change the viscosity of tration in the range 0.0–1.0 mM with a correlation coefficient
54
2
the DNA. The results of these two methods strongly suggest that value R = 0.99816 (Fig. 3b). According to the IUPAC convention,
the mode of binding of the synthesized alkaloid to CT-DNA was the limit of detection value (3s/k) was 46 nM, which was
mainly groove binding (Fig. S2a and b, ESI†). Details regarding the comparable and superior to previously reported work (Table 1)
2ꢀ
mode of binding of BER-S to DNA are described in the ESI.†
and much lower than the WHO-permitted S concentration in
drinking water. Thus, the BER-S/DNA complex could act as a
2
ꢀ
3
.5. Spectral recognition study of S by the BER-S/DNA
complex
.5.1. UV-vis spectroscopic recognition of S by the BER-S/
DNA complex. We studied the change in absorption spectra of values of S in the colorimetric method without DNA and in
2ꢀ
superior fluorescence turn-on template for the detection of S
.
2
ꢀ
3
3.5.2.2. Comparison and discussion of the two LODs. The LOD
2
ꢀ
2
ꢀ
BER-S/DNA complex in the presence of S . Upon DNA binding, the fluorometric method in the presence of DNA were 56 nM
the absorption maxima of BER-S shifted from 342 nm to and 46 nM, respectively. As the fluorometric method has a
3
S
47 nm (Fig. S10, ESI†), and thereafter, the introduction of lower LOD than the colorimetric method, we concluded that
2
ꢀ
elicited a red-shift from 347 nm to 368 nm, but in the BER-S in the presence of DNA could act as a better probe for the
2
ꢀ
presence of another anion, no perceptible spectral changes detection of trace S in real samples as well as in living cells.
were observed, which accordingly tunes the colorimetric detec- The detection of sulfides in the presence of DNA, i.e., in the cell,
2
ꢀ
tion of S by BER-S/DNA template (Fig. S11, ESI†). Thus, the is crucial as our body is easily contaminated by sulfide through
2ꢀ
BER-S/DNA complex could perform as an S detector probe.
drinking water as it is water soluble or through inhalation,
.5.2. Fluorescence spectroscopic recognition of S by the which damages the cell. With this study, we became aware
BER-S/DNA complex of the damaged cells due to sulfides compared to normal cells.
.5.2.1. Emission titration spectra. Further strong evidence In this platform, the sensing of sulfide in the presence of DNA
2ꢀ
3
3
2
ꢀ
for the detection of S by the BER-S/DNA template comes from is essential, and our synthesized analogue assured it could
fluorescence spectroscopy. BER-S/DNA complex exhibited a meet this purpose.
weak fluorescence (f = 0.0059) (Table S3, ESI†), but upon the
2
ꢀ
successive addition of S , the emission intensity at 530 nm
3.5.2.3. Selectivity and sensitivity. The excellent selectivity
2
ꢀ
increased gradually up to saturation (0 to 20 mM), and then after of the BER-S/DNA complex toward S
was examined by
saturation the intensity was almost 10-fold higher compared to measuring the change in emission intensity (I530) in the
2ꢀ
the complex (Fig. 3a), which made us consider that the BER-S/ presence of S and other relevant anions. Almost no change in
2
ꢀ
DNA complex is an excellent turn-on fluorescent sensor for S
.
emission intensity was seen with the addition of various anions as
2ꢀ
This is attributed to the fact the cleaved product berberrubine stated earlier, but surprisingly, upon the addition of S , a vast
also interacts with DNA (berberrubine–DNA complex, f = 0.02) increase in the fluorescence intensity at 530 nm was observed
(Table S3, ESI†), for which a remarkable change in fluorescence (Fig. S12, ESI†). Again, we checked the selectivity in the presence of
intensity was observed. Accordingly, we plotted the emission various cations and other life elements and no observable change
2ꢀ
intensity at 530 nm (I530) vs. concentration of S , which in fluorescence spectra was observed (Fig. S13 and S14, ESI†).
reflected a nonlinear S-like curve and consequently a good Additionally, we also examined the change in I530 in the
Fig. 3 (a) Fluorescence response of BER-S/DNA complex (probe = 10 mM, DNA = 150 mM) with increasing concentration of sulfide (0–2 equiv.).
(
l
b) Plot of fluorescence intensity at 530 nm of BER-S/DNA complex (probe = 10 mM, DNA = 150 mM) upon varied concentrations of sulfide (0–20 mM),
ex = 360 nm, lem = 530 nm, and the fl. intensity was linearly related to the concentration of sulfide (0.0–1.0 mM) in CP buffer solution (inset).
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Table 1 Comparison of some published sensor-system for the detection of sulfide anion
Sensor system
Method
Solvent system
Linearity range
Detection limit
Ref.
Bimetallic terbium(III)/copper(II) complex
A piperazine linker, an 8-aminoquinoline ligand
Fluorometric
Fluorometric
10 mM HEPES buffer
HEPES buffer/ethanol
0–2 mM
0.5–8 mM
130 nM
280 nM
18
19
(
6 : 4 v/v)
HEPES aqueous buffer
THF–H O = 3 : 7)
7
-Nitrobenz-2-oxa-1,3-diazole (NBD)
-Hydroxy-1-naphthaldehyde
Fluorometric
Colorimetric
0–3.0 mM
0.17 mM
21
22
(
2
2
Bis–tris buffer/DMSO
solution (v/v = 1 : 1)
Not mentioned
2.2 mM
Naphthalene diimide
Isophorone-based probe
dsDNA–CuNPs
Fluorometric
Fluorometric
Fluorometric
Colorimetric and
fluorometric
CH
3
CN
Not mentioned
0–200 mM
0–2 mM
8.7 mM
0.53 mM
80 nM
46 nM
40
46
50
This
work
100% PBS buffer
Aqueous
100% CP buffer
Berberine analogue
0–1.0 mM
presence of biological thiols, i.e., GSH, Cys, Hcy, Met, Cyt, and complex, bearing in mind if our probe could also be applicable in
TBA. However, no remarkable change was observed in the a biological system. Tiny changes in emission intensity (I530) of
2ꢀ
fluorescence spectra in the presence of this thiol (Fig. 4a).
BER-S/DNA complex in the presence of S were observed in the
The above result is consistent with our expectation that the biological relevant pH range 4–9 (Fig. 4b). This result indicates that
2
ꢀ
sulfonate linkage is selectively attacked by S only and not by the BER-S/DNA complex is stable in the above pH range for the
2
ꢀ
2ꢀ
other ions, and also due to the greater nucleophilicity of S
selective detection of S . Thus, we used pH 7.2 for the fluorometric
2ꢀ
compared with GSH, Cys, Hcy, Met, Cyt, and TBA. Thus the detection of S by the BER-S/DNA complex, which is a significant
above result indicates that the BER-S/DNA complex could pH for the biological application of the probe.
specifically detect S among the other environmentally and
biologically abundant anions.
2
ꢀ
3
.5.2.6. Time-dependent study. We also performed a time-
dependent kinetics study to scrutinize the time required for the
sensing response by the BER-S/DNA complex (10 mM). Upon the
3
.5.2.4. Competitive ion study. To scrutinize whether the
2ꢀ
addition of 2 equiv. S to the BER-S/DNA complex in CP buffer
solution (pH = 7.2), the fluorescence intensity at 530 nm no more
increased after 24 min (Fig. S17, ESI†). The above result indicates
sensitivity of the BER-S/DNA complex toward sulfide anions
could remain unchanged under the various potential competi-
tive anions, 2 equiv. of sulfide anions were added to the BER-S/
DNA (10 mM) in the presence of various relevant anions
2 equiv.) in pH 7.2 CP buffer solution (Fig. S15 and S16, ESI†).
As shown in Fig. 4a, various biothiols have no interfering effect
on the fluorometric detection of S . Thus, the complex could
perform as a good fluorescence turn-on S detector template
even involving various relevant potential anions and biothiols.
2
ꢀ
the complex could detect S after 24 min of its addition.
3.5.3. Proposed sensing mechanism study
(
3.5.3.1. In colorimetric sensing. On the basis of the above
2
ꢀ
sensing experiment, the sensing mechanism for the detection
2
ꢀ
2
ꢀ
of S
was logically explained by the nucleophilic attack
2
ꢀ
of S toward the sulfonate linkage to produce the cleaved
product berberrubine. For establishing the proposed sensing
3
.5.2.5. pH sensitivity. We further examined the effect of pH mechanism, we applied various analytical tools, like ESI-MS
2ꢀ
toward the fluorescence turn-on detection of S by the BER-S/DNA and IR spectroscopy. Further strong evidence came from the IR
2ꢀ
Fig. 4 (a) Selectivity and competitive study of BER-S + DNA complex in presence of biothiols (1: GSH, 2: Hcy, 3: Cys, 4: Met, 5: Cyt, 6: TBA, 7: S ). The blue bar
represents the I530 of BER-S/DNA complex, red bars represent the I530 after the addition of biothiols and the olive bars represent the I530 of complex after the
2ꢀ
2ꢀ
addition of S in the presence of biothiols. (b) Fluorescence intensity (I530) of BER-S/DNA complex and BER-S//DNA complex + S at various pH values.
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2
ꢀ
2ꢀ
Fig. 5 (A) (a) HRLC-MS spectrum of BER-S + S (1 : 1 equiv.), (b) BER-S + S (1 : 2 equiv.), and (c) cleaved product, BER-OH. (B) IR spectra of the probe
ꢀ1
BER-S (blue), upon the addition of sulfide (red), a broad band appears at 3202 cm assigned to the O–H stretching vibration. (C) In the presence of DNA,
the fluorescence titration of BER-OH (10 mM) with increasing concentration of CT-DNA (up to 150 mM), lex = 360 nm, lem = 530 nm.
spectral experiment. The IR spectra of BER-S after the addition of sulfide. Accordingly, when we carried out the fluorescence
2
ꢀ
ꢀ1
of 3 equivalent S showed a new broad peak at 3202 cm
which was assigned to OH vibration stretching frequency DNA, the maximum fluorescence intensity of BER-OH (Fig. 5C)
Fig. 5B), which further confirmed the generation of the hydroxyl was 10-fold greater than that of BER-S (Fig. 3a); comparable to that
,
titration of BER-S and BER-OH with increasing concentration of
(
group on berberine, i.e., berberrubine. Further confirmation of the of the maximum fluorescence intensity obtained in the sulfide
proposed mechanism came from the mass spectroscopic analysis. detection study (Fig. 3a). Thus, the above outcomes clarify the
2ꢀ
HRLC-MS of the solution containing BER-S/S
(1: 1 equiv.) mechanism of sulfide detection in the presence of DNA.
exhibited the two most abundant peaks with almost a 1 : 1 ratio
at m/z 552 and 321, which indicated that both BER-S (m/z = 552)
and the cleaved product BER-OH (m/z = 321) are present in solution
at this stage (Fig. 5A(a)). Consequently, if we performed the mass
3
.6. Lifetime measurement
The lifetime data were measured by the time-correlated single
photon counting (TCSPC) procedure using CP buffer of pH 7.2
at the excitation wavelength of 372 nm from a laser LED source.
The time-resolved lifetime decay profile, F(t) was studied by the
following equation:
2ꢀ
spectra of the solution containing BER-S and S (1 : 2.5 equiv.),
only one abundant peak at m/z 321 (Fig. 5A(b)) was observed,
corresponding to berberrubine (m/z = 321) (Fig. 5A(c)), i.e., at
this stage all BER-S molecules undergo nucleophilic reaction by
2
ꢀ
X
a
S
anion. Thus, the IR and HRLC-MS analysis successfully
FðtÞ ¼
ai expðꢀt=tiÞ
established our proposed sensing mechanism.
i¼1
3.5.3.2. In fluorometric sensing. The sensing mechanism is where n represents the number of segregate decay components,
quite helpful in the presence of CT-DNA, and we can explain a and t indicate an excited state fluorescence lifespan and the
i
i
the sensing mechanism as follows. In the absence of DNA, the pre-exponential factors correspondingly correlated by means of
6
0
2
cleaved product BER-OH was confirmed by various analytical the ith component. The chi-square (w ) value indicates the best
methods as stated previously. In the presence of DNA, the cleaved fitting. The consequential data were analyzed by EZ-time soft-
product BER-OH could also interact with DNA, which plays a ware. The TCSPC measurements were performed to realize the
decisive role in emission intensity enrichment upon the addition mechanism of the turn-on responses of the BER-S/DNA complex
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Fig. 6 (a) HOMO–LUMO energy level of probe BER-S and the corresponding product, BER-OH, (b) time-resolved fluorescence spectra of BER-S, BER-
2
ꢀ
S/DNA complex, and BER-S/DNA + S (2 equiv.), lex = 372 nm and lem = 530 nm.
2ꢀ
Table 2 Fluorescence lifetime data of BER-S, BER-S + DNA complex, and The product, after the addition of S anions, BER-OH, had a
2
ꢀ
BER-S + DNA complex + S (lex = 372 nm) at pH 7.2
lesser energy gap compared to BER-S, confirming the red-shift in
UV-vis spectra, which clarified our experimental results (red-shift
from 342 to 377 nm and 430 to 490 nm).
2
Entry
t
1
(ps)
a
1
t
2
(ns)
a
2
t
av (ns)
w
BER-S
BER-S + DNA-complex
418
806
0.78 4.05
0.67 2.48
0.61 2.06
0.22 1.217
0.33 1.358
0.39 1.192
1.18
1.17
1.11
3.8. Analysis of real samples
BER-S + DNA-complex + 637
2
ꢀ
2
.0 equiv. S
The desired application of the probe (BER-S) was investigated
2
ꢀ
by means of the quantitative detection of S in waste water,
2ꢀ
toward S ions (Fig. 6b). The average fluorescence lifetime (t) of tap water, and drinking water samples. The waste water sam-
BER-S in CP buffer was 1.2 ns, while the average fluorescence ples were collected from a spinning mill near our university
lifetimes of the BER-S/DNA complex and BER-S/DNA complex + campus and used as usual. The experimental results (Table 3)
2ꢀ
2ꢀ
S
were 1.36 ns and 1.19 ns, respectively (Table 2). The indicated that the probe could recognize the spiked S in real
fluorescence quantum yield of BER-S was 0.0005, whereas the samples and the detected concentration concurred well with
quantum yield of the BER-S/DNA complex was 0.006, and the added concentration, and the recoveries were acceptable
accordingly an increase in the fluorescence lifetime of BER-S too. This result revealed that the probe has proper exactness
2
ꢀ
upon binding with DNA was expected, but only a little increment and accuracy for a quantitative finding of S in water samples
in lifetime was observed. The BER-S had two exponential decay and has the potential to be used in water quality monitoring.
natures, and it was observed that the lifetime of the major
3
.9. Living cell imaging studies
contributing exponential decay of 65% increased upon binding with
DNA and that tuned the fluorescence intensity enhancement. Again,
with the introduction of S anions (2 equiv.) to the BER-S/DNA
complex, the fluorescence quantum yield increased from 0.006 to
To further demonstrate the biological imaging application of
BER-S in living cells, the cytotoxicity of the probe was evaluated
first by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] assay. The average value of four independent experiments
was taken, and it was found that the compound had an IC50 value of
2ꢀ
0.02, but the lifetime decreased (Table 2). This anomalous fluores-
cence property, i.e., an increase in quantum yield but decrease in
lifetime, may due to the enhanced local environment around the
BER-S/DNA complex and higher emission rate constant.
12.3 mM in A375 cells (Fig. S18, ESI†). Accordingly, we performed
61
Table 3 Detection of sulfide anions in real samples using the BER-S/DNA
complex as a fluorescent probe
3.7. Density functional theory (DFT) calculations
We further scrutinized the change in electronic structure and
observed the photophysical properties of BER-S upon the addi-
tion of sulfide anions via DFT study. The optimization of the
synthesized analogue BER-S and its deprotected form, BER-OH,
and DFT calculations were performed using the Gaussian 09
program at the CAM-B3LYP/6-31g(d,p) level. The resultant opti-
mized geometries of BER-S and BER-OH are depicted in Fig. S19,
ESI† and the frontier molecular orbital energies of HOMO and
LUMO of BER-S and BER-OH were also calculated (Table S4,
ESI†). The HOMO to LUMO energy gaps of BER-S and BER-OH
were calculated as 3.15 eV and 2.37 eV, respectively (Fig. 6a).
Na
(mM)
2
S added Na
2
S found
Recovery RSD
(%) (%, n = 3)
Samples
(mM)
Waste water
1.5
1.49 ꢄ 0.02
2.04 ꢄ 0.0264
3.0267 ꢄ 0.0472 100.88
99.33
97.14
1.34
1.29
1.58
2.1
3.0
Tap water
1.5
1.4633 ꢄ 0.0416
2.0767 ꢄ 0.0252
2.99 ꢄ 0.0964
97.33
98.57
99.66
2.84
1.21
3.22
2.1
3.0
Drinking water 1.5
.1
3.0
1.48 ꢄ 0.0173
98.66
1.17
0.72
1.21
2
2.1067 ꢄ 0.0153 100.32
2.98 ꢄ 0.0360
99.33
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Fig. 7 (A) Fluorescence microscopic images of skin melanoma cells treated with 3 mM BER-S for 30 min and thereafter treated with various
2
ꢀ
2ꢀ
concentrations of S . (B) Skin melanoma cells were treated with 6 mM BER-S for 30 min and then various concentrations of S . (C) Skin melanoma
2
ꢀ
BER-S were treated with 10 mM BER-S and then various concentrations of S for 30 min, 200ꢃ magnification.
concentration variant living cell imaging to examine whether the excellent selectivity and sensitivity over other analytes, includ-
probe could be used to detect sulfide in a biological system. The ing biothiols. Having no noticeable interference from other
4
fluorescence microscopic image of a cell culture (2 ꢃ 10 cells per analytes, including biothiols, makes the BER-S and BER-S/DNA
35 mm culture plate) containing 3 mM BER-S after 30 min incubation complex a proficient probe for sulfide detection. The limit of
showed no prominent fluorescence in the cells. After that, the cells detection of BER-S and BER-S/DNA complex was as low as
2ꢀ
were treated with various concentrations of S up to 2 equivalents, 46 nM and 56 nM, respectively, for the detection of sulfide
2
ꢀ
and the results indicated that with increasing the concentration of anions, which are much lower than the extreme level of S
S
2ꢀ
, there was an increasing strong red fluorescence in the cells (15 mM) in drinking water acceptable by the WHO. The reaction-
(Fig. 7A). Similarly, we performed the said experiment taking based sensing mechanism was well established via IR, HRMS,
another two concentrations of BER-S (6 mM and 10 mM) and with DFT, and fluorescence lifetime measurement. Real sample
increasing the sulfide concentration up to 2 equivalents in another analysis and a live cell imaging study indicated the probe could
2ꢀ
two set of samples, and we got strong, eye-catching red fluorescence be successfully used to detect S
in environmental water
increasingly (Fig. 7B and C). From the above concentration variant samples and living cells, respectively. Therefore, the BER-S
cell imaging experiment, we concluded that our probe BER-S with a and BER-S/DNA complex have potential applications in envir-
2
ꢀ
2ꢀ
higher concentration (6 or 10 mM) could detect S in living cells, onmental and biological systems to assay S and we deem that
and even a very low concentration of BER-S (3 mM) could detect a the results reported in this article could offer a new opportunity
lower concentration of sulfide in living skin melanoma cells effec- for designing and effortlessly synthesizing superb selective
2
ꢀ
tively. These results indicate that the probe had good cell perme- fluorescent chemodosimeters for S in the presence of DNA.
ability and the probe had a excellent capability for the fluorescence
imaging of sulfide anions in vivo.
Conflicts of interest
4
. Conclusions
There are no conflicts to declare.
We developed a new probe BER-S based on isoquinoline
alkaloid berberine with advantages of excellent water solubility.
The probe BER-S could act as a colorimetric chemodosimeter in
Acknowledgements
the absence of DNA and amazingly, as a fluorescent turn-on This study is financially assisted by the Department of Science
probe in the presence of DNA for aqueous sulfides and showed and Technology (DST) Science and Engineering Research
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NJC
Board (SERB), Govt. of India, No. SB/FT/CS-051/2013 (SERB/F/3694/ 22 S. M. Kim, M. Kang, I. Choi, J. J. Lee and C. Kim, New
015-2016) and G. C. Jana gratefully acknowledges CSIR for Senior
J. Chem., 2016, 40, 7768.
Research Fellowship (09/599(0058)/2014-EMR-1). M. Khatun and 23 C. Kar, M. D. Adhikari, A. Ramesh and G. Das, Inorg. Chem.,
Sk. Nayim thanks to UGC for MANF fellowship. We are also
2013, 52, 743.
obliged to USIC (Vidyasagar University), UGC-SAP DRS-II and 24 S. Wang, S. Xu, G. Hu, X. Bai, T. D. James and L. Wang, Anal.
2
DST-FIST New Delhi and for instrumental facilities.
Chem., 2016, 88, 1434.
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5 F. J. Huo, J. Kang, C. Yin, J. Chao and Y. Zhang, Sci. Rep.,
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6 J. Kang, F. Huo, P. Ning, X. Meng, J. Chao and C. Yin, Sens.
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7 Q. Zhao, J. Kang, Y. Wen, F. Huo, Y. Zhang and C. Yin,
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