A fluorescent probe for bisulfite ions: its application to two-photon tissue imaging

Article abstract of DOI:10.1039/C6TB02637K

A benzoxazinone based fluorescent probe for the specific and efficient detection of bisulfite ions in aqueous medium is described. The probe formed a bisulfite/sulphite adduct with an associated turn-on fluorescence response in the red wavelength region. No interference was observed in the detection process from all possible competing anions and molecules, including cyanide ion, cysteine, homocysteine and glutathione. In addition, the probe showed a fast response time, low detection limit, and cell membrane permeability. Furthermore, the probe was two-photon excitable, enabling imaging of endogenous bisulfite ions in HeLa cells as well as in deep tissues from different organs of mouse.

Full text of DOI:10.1039/C6TB02637K

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A Fluorescent Probe for Bisulfite Ion: Its Applications to Two-
Photon Tissue Imaging
maReceived 00th January 20xx,
Accepted 00th January 20xx
Hridesh Agarwalla,a Suman Pal,a Anirban Paul,b Yong Woong Jun,c Juryang Bae,c Kyo Han Ahn,c*
Divesh N. Srivastava,b Amitava Dasa,b
*
DOI: 10.1039/x0xx00000x
www.rsc.org/
A benzoxazinone based fluorescent probe for specific and efficient detection of bisulfite ion in aqueous medium is
described. The probe formed a bisulfite/sulphite adduct with an associated turn-on fluorescence response in the red
wavelength region. No interference was observed in the detection process from all possible competing anions and
molecules, including cyanide ion, cysteine, homocysteine and glutathione. In addition, the probe showed a fast response
time, a low detection limit, cell membrane permeability. Furthermore, the probe was two photon excitable,enabling
imaging of endogenous bisulfite ion in HeLa cells as well as in deep tissue from different organs of mouse.
body weight.18 US Food and Drug Administration (FDA) allows
Introduction
2
10 ppm (125ꢀ
is a need to develop appropriate methodology capable of
2
detecting SO2 or SO3 ˉ/HSO3ˉ present at low concentrations in
M) of SO3 ˉ in food and beverages.19 Thus, there
Anions are ubiquitous in nature and participate in various
critical chemical and biological processes. Significance or
implication of such processes in the environment or in living
organism has received increasing interest in the development
of new sensing systems for specific detection and
quantification of such anions.1-3 With the advent of industry in
modern era, sulfur dioxide (SO2) becomes an inevitable air
pollutant originating from the combustion of fossil fuels and
coal, as this has deleterious influences on the environment and
human health.4-5 On inhalation of SO2, it is hydrated in
respiratory tract to produce sulfite and bisulfite species, which
are actually responsible for symptoms of urticaria,
hypotension, diarrhea, and dermatitis.6-10 Bisulfite can also
cause allergic reaction, breathing problems, wheezing, hives
and gastrointestinal pain.11-12 Despite their adverse influences
on human health and environment, SO2 and its hydrated forms
are widely used for their antioxidant, antimicrobial and
preservative property.13-15 SO2 is frequently used in wine
industry for preventing bacterial growth as well as in
pharmaceutical industry for preventing oxidative degradation
during storage.16-17 Considering its adverse influences while
using in food, beverage and pharmaceutical industry,
FAO/WHO have jointly suggested that the acceptable daily of
intake of SO2 for a healthy human should not exceed 0.7 mg/kg
food/pharmaceutical products and also the environment.
Many methods have been developed for quantification of
sulfites/bisulfites in food and beverages such as Monier-
Williams
method,20
spectrophotometry,21-22
capillary
electrophoresis,23-24 and chromatography.25-26 However, such
methodologies are mostly time consuming and involve special
sample preparation, limiting their use in field sensor
application and in imaging application. High enthalpy of
2
hydration further makes the sensing of SO3 ˉ/HSO3ˉ in
aqueous media
a challenging issue. Albeit it has been
demonstrated more recently, the chemodosimetric detection
process involving higher enthalpy of reaction could be a
preferred methodology for designing an efficient molecular
probe for such anions.27-28 Fluorescence-based process is
generally preferred for designing chemodosimetric reagent for
specific recognition of these SO2 derivatives. This is because of
higher sensitivity in the detection process as well as the
possibility of using such reagent for imaging application in
mapping the distribution of intracellular sulfite/bisulfite
species in live cells/tissue.29-31 Most of the fluorescence-based
probes developed for the detection of bisulfite ion are based
on the nucleophilic addition reaction with aldehyde,32-37
deprotection of levulinate group,38-40 interaction with amine41
or Michael type addition reaction.42-49 However, these probes
generally suffer from long response time, interference from
biothiols (cysteine, homocysteine, glutathione, or aminothiols)
or protease/esterase, or occasionally from low detection limit.
Thus there is a need for a sensitive and specific probe with fast
response time and the above referred limitations alleviated.
Significance of such a probe is more if it allows luminescence
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response in the red region of the spectrum—as this helps to spectra were recorded using a Perkin Elmer Lambda 950 UV-
reduce interference due to autofluorescence from intrinsic Vis spectrophotometer equipped with a quartz cuvette (path
biomolecules, which generally occur in the shorter length of 1cm). Fluorescence spectra were recorded on a PTI
wavelengths. Furthermore, it is more advantageous if such a QuantaMaster 400 spectrophotometer. ESI-Ms measurements
probe can be used for two-photon imaging in the near-infrared were carried out on
a Waters QTof-Micro instrument.
wavelength region (~ 900 nm or so), which causes low Microanalyses (C, H, and N) have been performed using a
phototoxcity, photobleaching, and autofluorescence while Perkin-Elmer 4100 elemental analyzer. The open circuit
offering high spatial resolution in deep tissue imaging.50-51 potentials were measured on source meter unit (SMU)
There are a few reports on two-photon microscopy (TPM)- [Keithley 2635A].
2
based probes for SO3 ˉ/HSO3ˉ, but most of these probes either
Synthesis of probe 1
emit in the blue/green region or require an exogenous source
of bisulfite ion for imaging. Furthermore, the possibility of
using such reagents in tissue imaging is not explored.52-56
Herein we report a new benzoxazinone derivative as a novel
fluorescent probe for bisulfite ion, which has pendant
aldehyde functionality in conjugation with a dimethyl amino
group for a pronounced intramolecular charge transfer effect.
This probe is found to be selective in reacting with bisulfite ion
in an aqueous medium in presence of all other possible
interfering reagents (especially cyanide ion and biothiols like
cysteine, homocysteine and glutathione) and shows turn-on
type luminescence response. The probe also shows relatively
short response time, low detection limit and significant
emission at longer wavelength (> 600nm), which allowed
chemodosimetric detection of bisulfite ion in an aqueous
medium as well as in biological objects. In addition, the
bisulfite adduct of probe was two-photon excitable and thus
allowed imaging of endogenous bisulfite ion in cells as well as
in tissues by TPM.
Synthesis of probe
benzo[b][1,4]oxazin-3-yl)vinyl}benzaldehyde, is shown in
Scheme 1. The benzoxazinone intermediate, 7-
1, 4-{(1E)-2-(7-(dimethylamino)-2-oxo-2H-
(dimethylamino)-3-methyl-2H-benzo[b][1,4]oxazin-2-one, was
synthesized by following the previous reports.57 these were
utilized for the synthesis of probe
benzoxazinone intermediate was suspended in 3.5 mL of acetic
anhydride. To this suspension, 0.54 (4.04 mmol) of
terephthalaldehyde was added and temperature and the
reaction mixture were maintained at 140 C for 4 h. The
1. 0.55 g (2.69 mmol) of the
g
°
mixture was then cooled to room temperature and a red
precipitate appeared. This was filtered and the residue was
thoroughly washed with cold diethyl ether to get the desired
product in pure form. No further purification was required and
an isolated yield of probe was 360 mg (41%). 1H NMR (500
MHz, CDCl3, 25oC, TMS): 10.0 (s, 1H, HCHO), 8.0 (d, 1H, J=15 Hz,
CH=CH), 7.9 (d, 2H, J= 8 Hz, C6H4), 7.8 (d, 2H, J=8 Hz, C6H4),
7.59 (d, 1H, J = 15 Hz, CH=CH),7.6 (d, 1H, J = 8.5 Hz, C6H3), 6.8
(dd, 1H, J= 6.5 Hz, 2.5 Hz, C6H3), 6.5 (s, 1H, C6H3), 3.1 (s, 6H,
N(CH3)2). 13C NMR (100 MHz, CDCl3, 25 oC, TMS): 214.5, 167.6,
165.5, 10.8, 153.3, 151.9, 145.1, 143.1, 137.8, 134.9, 132.1,
Experimental section
129.9, 113.0, 96.5, 25.5 IR (KBr) ν
1382. ESI Ms (m/z): 318.80 [M
max/cm−1: 1718, 1688, 1609,
H+]. Elemental analysis:
Materials and Methods
ꢀ
ꢀ
The benzoxazinone intermediate, used for the synthesis of
C19H16N2O3 calculated C (71.24), H (5.03), N (8.74); found C
(71.5), H (4.97), N (8.8).
probe 1, was synthesized by adopting a previously reported
procedure.57 Terephthalaldehyde, ethyl pyruvate, Pd-charcoal
(10%) were purchased from Sigma-Aldrich and were used as
received. Acetic anhydride, dimethyl sulfoxide (DMSO, HPLC
Preparation of stock solutions
grade), NaNO2, HCl, hydrazine hydrate, ethanol, selenium A solution of probe
1 (1 mM in DMSO) was prepared and used
dioxide, 1,4-dioxane and salts of different anions were of for all experiments after appropriate dilution with a buffer
reagent grade (S. D. Fine Chemical, India) and used as solution of Na2HPO4:citric acid (200 mM, pH 5.0) to make a
5
received. Solution pH was evaluated using a Mettler Toledo final concentration of
1
as 2.0
×
10− M. Stock solutions (100
FEP20 pH meter. FTIR spectra were recorded as KBr pellets in a mM) of different anions (sodium salts of Fˉ, Clˉ, Brˉ, Iˉ, NO3ˉ,
cell fitted with a KBr window, using a Perkin-Elmer Spectra GX SO42ꢁ, OAcˉ, CNˉ, S2O32ꢁ, S2ˉ, N3ˉ, HSO3ꢁ, SO32ꢁ, and H2PO4ꢁ)as
2000 spectrometer. 1H and 13C NMR spectra were recorded on well as solutions of cysteine (Cys), homocysteine (Hcy), and
a Bruker 200/500 MHz FT NMR (Model: Avance-DPX 200/500) glutathione (GSH) were prepared in the Na2HPO4:citric acid
using trimethylsilane (TMS) as an internal standard. Absorption buffer solution (200 mM, pH 5.0). Stock solutions were further
diluted with the buffer solution as per requirement.
Cell imaging
HeLa human cervical cancer cells were obtained from Korean
Cell Line Bank. HeLa cells were incubated in DMEM
supplemented with 10% (v/v) fetal bovine serum (FBS) and 1%
Scheme 1. Synthesis of probe 1.
(w/v) penicillin-streptomycin (PS) at 37 °C in a humidified
atmosphere of 5% of CO2 in the air. Cells were passaged when
2 | J. Name., 2012, 00, 1-3
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they reached approximately 80% confluence. Cells were were immersed in 4% aqueous paraformaldehyde for one
seeded onto a cell culture dish at a density of 1.0
105 cells, week to fix the tissue and the other tissues were used without
which was incubated at 37 C overnight under 5% CO2 in the the fixation process. After the fixation, the tissues were
air. For imaging of endogenous bisulfite in cells, the cells in immersed in the probe solution (10 μM) for 30 min at 37 C.
×
°
°
DMEM were incubated with the probe (10 μM) for 30 min and The stained samples were placed on a slide glass for imaging,
then subjected to imaging by two-photon microscopy (TPM). after washing with PBS three to four times. Experimental set-
Both negative and positive control experiments were also ups for tissue imaging were essentially the same as described
conducted. For the negative control experiment, the cells were above for the cellular imaging, except for the emission light
pre-incubated in DMEM containing formaldehyde (1 mM) for collection both at 500 – 550 nm and 565 – 605 nm. The tissue
30 min, washed with PBS (phosphate buffered saline) buffer slice samples were mounted on a tight-fitting holder. The
for three times, and then incubated with the probe (10 μM) for excitation laser power was approximately 24.2 mW at the focal
30 min. For the positive control experiment, the cells were point, and the image depth was 80–100μm from the surface.
incubated in DMEM containing sodium bisulfite (1 mM) for 15 The imaging resolution was 1024 × 1024 pixels and the
min, washed with PBS buffer solution for three times, and then scanning speed was 200 Hz during the entire imaging. The
incubated with the probe (10 μM) for 30 min (ESI figure S16). acquired images were processed using LAS AF Lite (Leica,
Those cells incubated finally with probe were washed with PBS Germany).
buffer three times and then fixed with 4% formaldehyde
solution for the microscopic imaging. Co-localization
Spectrophotometric studies
experiments were conducted similarly: the cells were
100
ꢀL of stock solution of probe 1 (1 mM in DMSO) was
incubated in DMEM containing 5 μM of the probe and then
further incubated with 500 nM of LysoTracker Deep Red for 30
diluted to 5 mL with 200 mM Na2HPO4:citric acid buffer
solution (pH 5.0). 100 molar equivalents of different anions
and biothiols were introduced to the probe solution to make a
final volume of 5 mL. Fluorescence spectra were recorded at
room temperature with excitation at 490 nm. Similarly,
titration experiment with HSO3ˉwas also done with gradual
increase of HSO3ˉ concentration at room temperature.
min at 37 °C under 5% CO2 in the air. After the incubation, the
cells were washed with PBS buffer three times to remove the
remaining probe and then fixed with 4% formaldehyde
solution. TPM imaging was performed using a Ti-Sapphire laser
(Chameleon Vision II, Coherent) at 140 fs pulse width and 80
MHz pulse repetition rate (TCS SP5 II, Leica, Germany) and a
40× objective lens (obj. HCX PL APO 40×1.10 W CORR CS,
506341, Leica, Germany). The two-photon excitation
wavelength was tuned to 880 nm for the probe. Each emission
light was spectrally resolved into multi-channels (λem = 500 -
550 nm, 565 - 605 nm) for the probe. The cells prepared as
above were mounted on a tight-fitting holder. The excitation
laser power was approximately 24.2 mW. The images were
consisted of 1024 × 1024 pixels, and the scanning speed was
maintained as 200 MHz. Cellular imaging by confocal
microscopy was also performed using Leica TCS SP5 II
Advanced System for LysoTracker Deep Red. This microscope
was equipped with multiple visible laser lines (405, 458, 476,
488, 496, 514, 561, 594, and 633 nm). For this one-photon
imaging experiment, 633 nm laser line and an emission filter
ranging from 660 to 700 nm were used. Acquired images were
processed using LAS AF Lite (Leica, Germany), and all the
images were covered with specific pseudo-colours: orange to
red (probe) and cyan (LysoTracker Deep Red).
Results and Discussion
Probe
1 was synthesized in moderate yield following a general
one step condensation reaction between the benzoxazinone
intermediate and terephthalaldehyde (scheme 1). The probe
was characterized by spectroscopic (1H, 13C NMR and ESI-Ms)
and elemental analysis. All such data confirmed the desired
purity of the probe. The probe was soluble in an essentially
aqueous buffer medium (98:2 aq. buffer:DMSO, v/v) using 200
mM Na2HPO4:citric acid buffer (pH 5.0) solution. This solution
was used for all other studies, unless mentioned otherwise.
Absorption spectra recorded for probe
1
showed a
π‒π*
-dimethyl
transition and a charge transfer transition with N,N
′
benzooxazinone moiety as the donor fragment at 325 and 445
nm, respectively. A shoulder is observed at ~ 490 nm, which
could also be attributed to ICT-based process (ESI Figure S5).
This was further confirmed from the shift of the absorption
([λmaxAbs] ~ 480 nm in toluene) and emission bands ([λmaxEms] ~
555 nm in toluene) to longer wavelengths by ~ 20 nm and ~ 45
nm, respectively, on changing the solvent polarity from
Tissue imaging
toluene to DMSO (ESI figure S6, S7). Luminescence of probe
was essentially quenched possibly due to the non-radiative,
lowest energy transition of n– * from the formyl group.
1
A Balb/C type mouse (6 weeks, female) was used for this
experiment. Basically experiments were done under light
protected conditions (in a dark-room and using aluminum foil).
The mouse was dissected after dislocation of the cervical
vertebra. Blood perfusion with phosphate buffered saline (PBS,
1X solution) was performed for elimination of blood. The
organs were dissected, washed with PBS buffer, and then
sliced with a vibrating blade microtome (VT1000S, Leica,
Germany) to 400 μm thickness. The brain tissue slice samples
π
Luminescence spectra of the probe remained unaltered when
recorded in the presence of all other anions (Fˉ, Clˉ, Brˉ, Iˉ,
2
2
2
SO3 ˉ, and
NO3ˉ, SO4 ˉ, OAcˉ, CNˉ, S2O3 ˉ, S2ˉ, N3ˉ, HSO3ˉ
,
and biothiols (Cys, Hcy, and GSH), except
H2PO4ˉ,)
sulfite/bisulfite (depending on the media pH). A switch-on type
luminescence response was evident when spectrum for the
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probe was recorded in the presence of either of these two
species (Figure 1a).
The quantum yield (
Φ
) for the probe
10− to 0.03 (75-fold) on incubation with 100
equivalent of HSO3ˉ for 5 min. Respective value was
evaluated using coumarin-6 ( = 0.78 in ethanol) as reference.
1 was found to increase
Figure 1. (a) Luminescence spectra for probe
1
(20
ꢀ
M) in absence and presence
4
of various ions (Fˉ, Clˉ, Brˉ, Iˉ, NO3ˉ, SO42ꢁ, OAcˉ, CNˉ, S2O32ꢁ, S2ˉ, N3ˉ, HSO3ˉ
,
from 4.3
×
SO32ꢁ
luminescence spectra for the probe (20
8.0
10-3 M) using λex of 490 nm. (c) Plot of kobs vs. [HSO3ˉ] for evaluating the
,
and H2PO4
ꢁ
)
and biothiols (Cys, Hcy, and GSH); (b) Changes in
Φ
ꢀ
M) in presence of varying [HSO3ˉ] (0–
Φ
×
overall rate constant for the reaction between the probe and HSO3ˉ. All studies
were performed in aq. buffer:DMSO (98:2, v/v) using 200 mM Na2HPO4:citric
acid buffer solution at pH 5.0 (λex/λem: 490/600nm).
Interestingly, broad emission spectrum having a maximum at ~
600 nm appeared with tail of the emission band extended
beyond 700 nm (Figure 1a). This luminescence response
formed the basis of using this probe for specific detection of
1.02
2 Only
(a)
(b)
0.96
0.90
0.84
2
-
2 + HSO
b
3
SO3 ˉ/HSO3ˉ in aq. buffer medium. Interference studies in
1718
presence of large excess (200 molar equiv.) of various anions
and biothiols were performed and the results clearly revealed
that all such analytes failed to interfere with detection of
1688
1609
1500
2500
2000
1000
1.0
0.8
0.6
2 only
a
-
2 + HSO
3
2
SO3 ˉ/HSO3ˉ (ESI figure S8).
1726
1613
1500
As discussed above, sulphite exists solely as bisulfite ion at pH
2
10
9
8
δ
7
ppm
6
5
2500
2000
1000
5.0 and as an equilibrium mixture of SO3 ˉ & HSO3ˉ at pH 7.2.58
Wavenumber (cm-1)
Figure 2. (a) Partial 1H NMR spectra of 1 in DMSO-d6. (b) IR spectra obtained in
absence and presence of 10 equivalents of HSO3ˉ
pH-dependent fluorescence spectra of
1 was measured in the
absence and presence of HSO3ˉ. No significant change in
fluorescence spectra was evident for the pH range of 3 - 9.
However, in the presence of HSO3ˉ, distinct luminescence
turn-on response was observed for the pH range of 4 - 6 and
such change was the maximum at pH ~ 5. Relative changes in
intensity as a function of time was found to be much more as
well as found to reach the respective plateau much faster for a
solution at pH 5 (about two times faster) than that was
observed for a solution at pH 7. This clearly revealed an
efficient reaction and much better response at pH 5 (ESI Figure
S9, S10). Hence, pH 5.0 was chosen as preferred one, and at
this pH bisulfite (pKa ~6.97) is solely the reactive species.
Significantly, this value is lower than the permitted level set by
WHO, FAO and FDA in environmental, food and
pharmaceutical samples, respectively. All these data confirmed
that the present chemodosimetric probe helped us in sensing
2
and quantitative estimation of SO3 ˉ/HSO3ˉ in an essentially
aqueous buffer medium without any interference from all
possible anions (including cyanide) and biothiols.
The proposed reaction between probe
studied by 1H NMR, IR and ESI-Ms spectroscopy. 1H NMR
spectra of showed a signal at 10.0 ppm (Ha) for HCHO in
1 and HSO3ˉwas also
1
DMSO-d6 (Figure 2a). On reaction with HSO3ˉ, a new peak
appeared at 4.99 ppm (Hb) with concomitant disappearance of
the signal for HCHO at 10.0 ppm (Figure 2a). This confirmed the
formation of the proposed adduct. Other aromatic protons
also showed an obvious upfield shift compared to those of
Further, fluorescence spectra of
1 (20 ꢀM) were recorded with
gradual increase in [HSO3ˉ] (Figure 1b). With increasing
concentration of HSO3ˉ, increase in luminescence intensity (λex
= 490 nm) was observed and this reached maximum at 8 mM
of HSO3ˉ. All fluorescence spectra were collected after 10 min
ensuring completion of reaction. Relative changes in
luminescence intensities as a function of time for a specific
concentration of HSO3ˉ helped us to evaluate the observed
pseudo first order rate constant (kobs) for the reaction between
probe
1 due to the lowering of the extent of conjugation and
the electron withdrawing influence. An IR signal responsible
for the aldehyde functionality for probe 1 appeared at 1688
cm-1, which completely disappeared on formation of the
bisulfite adduct (Figure 2b). A distinct signal at m/z of 402.69
(1
+ HSO3ˉ + H+) with anticipated isotope distribution further
1
and HSO3ˉ at pH 5 (ESI figure 11). A linear dependency of kobs
as a function of [HSO3ˉ] (kobs = kc [HSO3ˉ] + c, where kc: rate
corroborated the proposed adduct formation (ESI Figure S13).
constant for the overall reaction and cis an intercept; Figure
1
1c) yielded kc of (18.9
±
0.3) s− (Figure 1c). These results
Two-photon imaging
confirmed that the luminescence enhancement involved
HSO3ˉ in the slow step of the reaction. The results clearly
revealed that relative changes in luminescence reached a
plateau within 4.5 min and offered the possibility of the rapid
hydrogen sulfite detection in an essentially aqueous buffer
medium (ESI Figure S10). An excellent linear relationship was
Probe
1 and its bisulfite adduct have dipolar character owing
to the presence of a donor (the amino group) and an acceptor
(the formyl/lactone moiety). As such dipolar dyes generally
have good two-photon excitable properties, we evaluated
their two-photon absorbing property. The bisulfite adduct of
observed for luminescence responses of probe
1 with varying
probe
1 showed a marginal but sufficient two-photon action
concentration of HSO3ˉ from 0 to 200 ꢀM), from which a
(
cross section (TPACS) value: the maximum two-photon action
cross-section value was determined to be 3.9 GM at 900 nm
(ESI Figure S14). On the basis of the two-photon fluorescent
data of the adduct, TPM imaging of bisulfite in cells and tissues
6
lowest detection limit of 1.86
Figure S12).
×
10− M was obtained (ESI
was performed. Probe
1 was found to be substantially toxic to
-
(a)
(b)
[HSO3 ] = 0 - 8.0 x10-3
M
(c)
0.025
6.00x105
4.50x105
3.00x105
1.50x105
HSO3
6.0x105
4.0x105
2.0x105
live Hct116 cells determined by MTT assay (ESI Figure 15) and
used further for imaging studies.
-
-
0.020
0.015
0.010
SO3
4 | J.0.N00ame., 2012, 00, 1-30.0
This journal is © The Royal Society of Chemistry 20xx
5.0x10-4 1.0x10-3
[HSO3-]/M
525
600
675
750
500
600
700
Wavelength (nm)
Wavelength (nm)
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TPM imaging of HeLa cells with probe
1 alone shows bright, Hydrolytic enzymes within the lysosome are activated by the
yellow-orange fluorescence, indicative of its imaging capability highly acidic pH (4.5 - 5.0) in the organelles interior.63
of endogenous cellular bisulfite.47,59-60 Cells pre-treated with Lysosomes generate and maintain their pH gradients by using
bisulfite (1 mM) (the positive control) show an increase in the activity of a proton-pumping V-type ATPase. As discussed
emission compared with that treated only with probe
1, which earlier, the rate of our chemodosimetric reaction and the
indicates that our probe senses bisulfite in cells (Figure 3a, ESI extent of emission intensity enhancement were higher for
2
figure S16).
HSO3ˉ than that of SO3 ˉ, and the former species exists
predominantly at pH ~ 5.0. This has presumably caused a
faster response to give the brighter images of lysosomes over
the dim background signal of cytosol where pH is around 7.4.
Figure 3: Fluorescence imaging of HeLa cells by TPM. (a) From the left: cells pre-
incubated with formaldehyde (1 mM) followed by of probe
incubated only with probe (10μM), and cells pre-incubated with 1.0 mM of
bisulfite followed by probe (10μM); (b) relative fluorescence intensity of
images of (a); (c) cells co-incubated with probe (5μM) and LysoTracker Deep
1 (10μM), cells
1
1
1
Red (500 nM): from the left, fluorescence collected from the probe channel
(500–550 nm and 565–605 nm) under two-photon excitation at 880 nm,
fluorescence collected from a LysoTracker channel (660–700 nm) under one-
photon excitation by 688 nm; (d) fluorescence intensity changes along with the
line of interest (ROI) in Figure c images (marked with blue and red solid lines).
Calculated colocalization factors are PCC = 0.628 and MOC = 0.897. The scale bar
is 25 μm.
A negative control experiment was also conducted in the
presence of formaldehyde, which is known to consume
bisulfite by forming an addition product. Indeed, the
Figure 4. TPM tissue imaging data: (a) Fluorescence images of five different
organs (brain, liver, lung, spleen and kidney); (b) Images of three different
hippocampus regions of mouse brain (DG, CA3 and CA1). All of the tissues were
fluorescence spectra of probe
1 did not show any emission
incubated with 10 μM of probe
1 and images were obtained after 30 min
incubation. The TPM imaging was conducted by exciting at 880 nm with 24.2 W
laser power at the focal point and by collecting fluorescence emission through
two overlaid channels (500 - 550 nm and 565 - 605 nm). Scale bar: 50 μm (in a),
75 μm (1st row in b), and 50 μm (2nd row in b). The depth of the tissue image is
around 80–100 μm from the surface.
enhancement when sample was pre-treated with
formaldehyde (ESI Figure S17). As expected, the cells pre-
incubated with formaldehyde did not show any notable
emission (Figure 3a), which confirms that bright emission was
due to presence of endogenous bisulfite. Endogenous sulfite is
known to be generated during the normal metabolism of
sulfur-containing amino acids, and alterations in sulfur-
containing amino acid metabolism occur in some
neurodegenerative diseases. Sulfite is also an important
intermediate in the metabolic pathway by which sulfur-
containing amino acids is converted to the corresponding
inorganic sulfate.61-62
Given that the probe stains endogenous cellular bisulfite
efficiently, next we move to ex vivo imaging of bisulfite in
organ tissues by TPM. For this purpose, five different organs of
mouse (brain, liver, lung, spleen and kidney) were prepared,
which were incubated with probe
1 (10μM) to sense
endogenous bisulfite. The TPM image data given in Figure 4a
clearly show bright fluorescence (with particle-wise brighter
fluorescence) in brain, liver and lung tissues, suggestive of a
high level of endogenous bisulfite in these organs and a very
low level in spleen and kidney. As the image data is intensity
based ones, at present it is difficult to estimate the probe
penetration/ distribution dependent on organs as well as the
bisulfite concentration. A further look into bisulfite in the
hippocampus region of brain, however, shows the presence of
bisulfite in all of DG, CA1 and CA3 areas where neurons are
located densely (Figure 4b). Furthermore, along the neuronal
pathway it also maintains particle-wise shape as does in the
cellular level (yellow arrows in figure 4b). The results
A closer look at the patterns in images shown in Figure 3b
clearly reveals that there are brighter particle-like spots over
the dim background. By analyzing the patterns of the
colocalization imaging with LysoTracker Deep Red,
a
commercially available lysosome specific dye, it is revealed
that the brighter particles are overlaid with the distribution of
lysosomes (Figures 3c
& 3d). Lysosomes, the terminal
organelles on the endocytic pathway, digest macromolecules
and make their components available to the cell as nutrients.
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DOI: 10.1039/C6TB02637K
ARTICLE
Journal Name
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detect bisulfite ion in an aqueous medium with turn-on type
fluorescence response in the red wavelength region. The
probe responds to bisulfite very fast with no interference from
common interfering agents such as biothiols and cyanide ion.
This emission on response was further utilized as an imaging
reagent for cellular uptake of bisulfite. Furthermore, as the
probe molecule is two-photon excitable, it was able to observe
endogenous bisulfite not only in cells but also in different
organ tissues with high resolution images of distinct
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A. D. acknowledges SERB (India) Grant no. SB/S1/IC-23/2013
and CSIR-CSMCRI Network project CSC0134 for funding. H. A.
and A. P. acknowledge CSIR for their research fellowships. S. P
acknowledges SERB Fund through Fast Track project. A.K.H.
acknowledges the financial support from Global Research
Laboratory Program (2014K1A1A2064569) through the
National Research Foundation (NRF) funded by Ministry of
Science, ICT & Future Planning in Korea. We thank Nandraj
Taye of National Centre for Cell Science, Pune for MTT assay.
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Journal of Materials Chemistry B
Page 8 of 8
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DOI: 10.1039/C6TB02637K
Graphical Abstract:
A Fluorescent Probe for Bisulfite Ion: Its Applications to Two-Photon Tissue
Imaging
Hridesh Agarwalla, Suman Pal, Anirban Paul, Jun Yongwoong, Bae Juryang, Kyo Han Ahn,
Divesh N. Srivastava and Amitava Das
DG
CA1
CA3
Two photon imaging of endogenous bisulphite of the hippocampus regions of mouse brain.