A mitochondria-targeted colorimetric and ratiometric fluorescent probe for biological SO2 derivatives in living cells

Article abstract of DOI:10.1039/c5cc03055b

A ratiometric fluorescent probe for SO₂ derivatives based on the conjugate of carbazole and indolium was presented, which could selectively respond to HSO₃^- over other thiol compounds. More importantly, CZ-Id is a novel mitochondria-targeted ratiometric fluorescent probe to image exogenous SO₂ derivatives.

Full text of DOI:10.1039/c5cc03055b

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A Mitochondria-targeted Colorimetric and Ratiometric
Fluorescent Probe for Biological SO2 Derivatives in
Living Cells†
Cite this: DOI: 10.1039/x0xx00000x
Yu Liu,a Kun Li,*ab Ming‐Yu Wu,a Yan‐Hong Liu,a Yong‐Mei Xie*b and Xiao‐Qi Yu*a
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
www.rsc.org/
A ratiometric fluorescent probe for SO2 derivatives based on manuscript, Yuan and Chang reported the first mitochondria-
the conjugate of carbazole and indoliumm was presented, targetable ratiommetric fluorescent probe for SO2 derivatives.10
which could selectively respond to HSO3‾ over other thiol
compounds. More importantly, CZ-Id is
mitochondria-targeted ratiometric fluorescent probe to image group also presented a water-soluble near-infrared probe for SO2
Recently, Guo group reported a ratiometric fluorescent probe for
6a
a
novel SO2 derivatives based on a near-infrared dye.
Not long ago, our
6b
exogenous SO2 derivatives.
and two mitochondria-targeted fluorescent probes for in vitro and in
vivo imaging ClO-.9 On the basis of above works, herein, we designed
two carbazole conjugates with indolium or pyridinium (Scheme 1).
The indolium or pyridinium unit here not only act as mitochondria-
targeted carriers but also improve the water solubility of the probe.
Sulfur dioxide (SO2) is considered to be a kind of air and
environmental pollutant for a long time. Epidemiological studies
have implied that SO2 exposure not only induces many respiratory
responses1 but is also linked to lung cancer, cardiovascular disease
and many neurological disorders.2 Therefore, the development of
analytical methods is highly needed for trace SO2 detection,
especially in those could realize detection in organisms. As an
excellent detection technique, fluorescent probes have attracted
increasing attention for their high selectivity and sensitivity as well as
real-time imaging. On the other hand, gas SO2 can be produced
endogenously from sulfur-containing amino acids and may regulate
vascular smooth muscle tone in synergy with NO.3 The role of SO2 in
tumour biology as well as in normal cells is still unclear. Therefore, a
quantitative detection, especially in situ detection of SO2 derivatives
in cells is of significant interest. Ratiometric fluorescent probes are
regarded as the potent tools for quantitative detection of various
analytes because they can eliminate most or all ambiguities by self-
calibration of two emission bands.4 In recent years, a number of
fluorescent probes have been reported for sulfite or bisulfite, while
only few ratiometeric fluorescence probes have been presented,
which provide a reliable method for quantity detection of SO2
derivatives.5,6 Since mitochondria are the main source of intracellular
ROS (reactive oxygen species) and RSS (reactive oxygen species),7
monitoring SO2 derivatives in mitochondria is particularly meaningful
Scheme 1. The structures of the probes.
The probes CZ-Id and PZ-Id could be easily prepared from the
condensation of 3-formyl-9-methyl carbazole with indolium or
pyridinium salt in the presence of piperidine with middle yields. The
target probes were characterized by 1H, 13C NMR and HRMS (see ESI).
Initially, we firstly tested the reactivity of the probes in different
solvents (ESI, Fig S1). When 10 equiv bisulfite was added to the CZ-Id
solution, the emission peak at 590 nm disappeared and a new
emission peak emerged at 490 nm, indicating that the probe CZ-Id
could respond to HSO3⎯ in a ratiometric mode. Meanwhile, CZ-Id
exhibited different reaction activities in different solvents. The
reaction could be completed in DMF solution almost in real time;
while it needs longer time in other solvents, and 5 min was necessary
for the reaction in PBS buffer solution. Considering the environment
within cells is rather homogeneous and slightly more hydrophobic
than in buffer,11 PBS/DMF mix medium (7:3, V/V) was selected as the
solvent for the following experiments (ESI, Fig S2). Disappointedly, no
obvious fluorescence change was found for PZ-Id at the same
and valuable. To date, quite
a lot of mitochondria-targeted
fluorescent probes have been developed,8, 9 However, to the best of
our knowledge, no example has been reported to quantify the
mitochondrial SO2 in living cells. While we are preparing the
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condition, which means PZ-Id could not respond to HSO3⎯ (ESI, intensity ratios to the concentration of HSO3 in the range of 0-50 μM
Fig.S3). We suspected that the different distribution of electron cloud was disclosed. According to the titration profiles, the detection limits
in the two probes resulted in the different reactivity towards bisulfite.
Therefore, only CZ-Id was investigated for detail.
(
S/N = 3) toward bisulfite were calculated to be 0.15 μM for CZ-Id
View Article O(nRlin=e
DOI: 10.1039/C5CC03055B
0.999, Fig. S8).
Subsequently, the time dependent fluorescence changes of CZ-
Id with HSO3⎯ were investigated (ESI, Fig.S4). When 10 equiv bisulfite
was added, the emission peak at 590 nm decreased rapidly, with a
new emission peak at 490 nm emerging, and the reaction could be
completed within 1 min, suggesting that the probe has good
potential application for the real-time imaging. The ratio of the
emission intensity at the two different wavelengths ( 490 /I590) for CZ-Id
I
varied from 0.0383 to 3.8769 after complete conversion, which is a
101-fold increasement. Meanwhile, in UV spectrum, the peak at 487
nm decreased and a new peak at 290 nm appeared, accompanied by
a colour change from pink to colourless (Fig. S5 and S6), implying the
break of intramolecular conjugated structure in CZ-Id
.
To examine whether CZ-Id can selectively detect SO2 derivatives
among other bio-relevant ions, we then measured the fluorescence
spectra of CZ-Id in the presence of various anions including the
common thiol compounds (Fig. 1). The addition of 500 equiv
Fig. 2 Fluorescence titration spectra of CZ‐Id upon the addition of HSO3‾ (0–50
μM) in PBS (pH 7.4, 10 mM, containing 30% DMF). [CZ‐Id] = 10 μM, λex = 350 nm,
slit: 5 nm/5 nm. Inset: the titration curve plotted with the fluorescence intensity
ratio of (I490/I590) changes of CZ‐Id to HSO3‾.
representative anions (F-, Cl-, Br-, I-, NO2 , HCO3 , N3 , AcO-, C2O42-, SCN-,
-
-
-
HPO42-, SO42-) did not significantly change the fluorescence of CZ-Id
.
20 equiv of other common sulphur compounds including thiol
species (Hcy, Cys, GSH), and even 500 equiv Cys and GSH caused no
To further investigate the sensing mechanism, high-resolution mass
spectroscopy (Figure S10) was used to confirm the addition of the
emission changes of the probe. In contrast, when CZ-Id was treated
bisulfite to CZ-Id. The prominent peak at m/z 445.1586
2
with 4 equiv HSO3
⎯
or SO3
⎯
, a dramatic ratiometric fluorescence
corresponding to the product CZ-S was found in HRMS spectra,
implying the addition product. Additionally, 1H NMR spectrometry
(Figure S9) was also adopted to study the reaction process. The
proton shift of the double bond at 8.64 ppm upshifted to 4.70 ppm,
which indicated that the bisulfite was added to CZ-Id via 1,4
conjugate addition. These results suggested that the reaction
mechanism was the same as that reported.6a-c, g
response was observed. Meanwhile, a synthetic SO2 donor, 6b, 9 which
was utilized to release SO2 , could also cause fluorescent changes to
CZ-Id with similar results(ESI, Fig.S7).
Scheme 2 The proposed sensing mechanism.
A standard MTT assay in A549 cells showed that more than 80%
of cells still remained alive even though 4 μM CZ-Id was internalized
for 24 h (Figure S11), indicating the acceptable cytotoxicity of CZ-Id
.
In light of the outstanding cellular penetrability and biocompatibility
of CZ-Id, further biological application of cell imaging was expanded.
It is well known that cationic small molecules like
triphenylphosphonium moiety could entry into mitochondria and
interact with anionic species via electrostatic interaction.12 Since
indolium is also a cationic moiety, we suspect it would exhibit the
Fig. 1 Fluorescence ratio of CZ‐Id (10 μM) in PBS (pH 7.4, 10 mM, containg 30%
DMF) to various anions
500 equiv. for Cys, Hcy and GSH, 4 equiv.for HSO3
（
500 equiv.
）
, and reactive sulphur (20 equiv. for S2‐,
2
⎯, 4 equiv. for SO3
⎯
), SO2
releasing agent (5 equiv. N‐benzyl‐2,4‐dinitrobenzenesulfonamide and 50 equiv.
Cys.). (for a: λex = 350 nm, λem = 490 nm; for b: λem = 590 nm).
same property. To prove the subcellular localization of CZ-Id
commercially available mitochondrial dye, Mito Tracker Deep Red
(MT DeepRed), was employed for co-localization study. As
,
Fluorescence titration experiments demonstrated that the
a
emission intensity ratios of the CZ-Id could be amplified evidently
upon addition of HSO3⎯
-, as depicted in Fig.2. The fluorescence of CZ-
Id could be and a linearly proportional increment of emission
illustrated in Fig. 3, the CZ-Id signal overlaid very well with the
fluorescence of MT DeepRed. The Pearson's co-localization
coefficient, that describe the correlation of the intensity distribution
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between two channels, was calculated to be 0.95, confirming that
CZ-Id were site-specifically internalized in mitochondria in living cells.
To the best of our knowledge, CZ-Id is the first ratiometric
fluorescent probe for detection of mitochondrial SO2 derivatives.
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DOI: 10.1039/C5CC03055B
Fig 3. HeLa cells were stained with CZ‐Id (2 μM) for 30 min, and then incubated
with MT DeepRed (1 μM) for another 20 min. (a): MT DeepRed; (b): CZ‐Id; (c):
merged images of (a) and (b), (d): bright field of (a); (e): intensity profile of ROIs
across HeLa cells. Red lines represent the intensity of CZ‐Id and blue lines
represent the intensity of Mito Tracker Deep Red. Ex@488 nm for red channel
(560‐640 nm), and Ex@638 nm for MT DeepRed (660‐720 nm). Bars: 10 μm.
Fig. 4 (a) Fluorescence imaging of HeLa cells incubated with CZ‐Id (2 μM) from
the green channel; (b) fluorescence imaging of (a) from the red channel; (c)
overlay of (a) and (b); (d) bright field of (a). (e) Fluorescence imaging of HeLa
cells incubated with CZ‐Id (2 μM) for 30 min and further incubated with NaHSO3
(20 μM) for 15 min from the green channel; (f) fluorescence imaging of (e) from
the red channel; (g) overlap of (e) and (f); (h) bright field of (e). (i) Fluorescence
c3imaging of HeLa cells incubated with CZ‐Id (2 μM) for 30 min and further
incubated with an SO2 donor (20 μM) and Cys (200 μM) for 15 min from the
green channel; (j) fluorescence imaging of (i) from the green channel; (k) overlap
of (i) and (j); (l) bright field of (i).Ex@405 nm for green channel (460‐520 nm),
Ex@488 nm for red channel (560‐640 nm). (m) Integarted emission intensity
Encouraged by the aforementioned results, we further
tested the quantity detection ability fluorescent probe in vitro. As
shown in Fig 4, a linearity ( 460-520/I560-620 versus HSO3⎯ concentration)
I
was established (Fig. 4m) via fluorescence titration, where I460-520 is
the integrated fluorescence intensity from 460 nm to 520 nm upon
excitation at 405 nm, and I560-620 is the integrated fluorescence
intensity from 560 nm to 620 nm upon excitation at 488 nm. Through
the good linear correlation, we could calculate the concentration of
unknown bisulfite sample. However, it is hardly to realize
quantification of mitochondrial SO2 concentration currently,10 due to
the low concentration of SO2 derivate existed in living cells. Therefore,
we try to quantify the mitochondrial SO2 derivatives in living cells by
ratios ( 460-520/I560-620) of CZ‐Id change as a function of HSO3‾ (0–100 uM) (n)
I
Statistical analysis were performed with student’s t‐test (n = 10 fields of cells).
***P < 0.001, **P < 0.01, error bars are ± sem.
Conclusions
In conclusion, a mitochondria-targeted, colorimetric and fluorescent
probe CZ-Id for SO2 in a ratiometric manner was reported, which
exhibits high sensitivity and excellent selectivity toward SO2 over Cys,
GSH and other anions with a rapid response time. This carbazole
indolium conjugates can penetrate cell membrane with low
cytotoxicity. In vitro imaging experiments show that CZ-Id can be
utilized to ratiometrically sense exogenous and endogenous SO2 in
mitochondria. We anticipate that this modular probe should prove
useful not only for detection of mitochondrial SO2 but also for
studying diseases associated with SO2 deviates and successive
research is ongoing.
treated with HSO3
⎯ and SO2 donor. As displayed in Fig. 4, adding 10
eq HSO3 or 10 eq SO2 donor will result in the increase of the
⎯
fluorescent ratio of green channel/red channel, suggesting it could
reflect the concentration of SO2 derivatives. By calculating the ratios
of CZ-Id in the presence/absence of HSO3
to the linearity in Fig. 4m, the average mitochondrial HSO3
⎯ in Fig. 4n and according
⎯
concentrations in HeLa cells were estimated to be 3.8 uM and 4.1 uM,
respectively. These resulted illustrated that only small amount of
HSO3
HSO3
⎯
⎯
entry into mitochondria, due to the wide dispersitation of
in cells. More importantly, it proved that CZ-Id could quantify
the concentration of SO2 derivatives in living cells.
Acknowledgement
This work was financially supported by the National Program on Key
Basic Research Project of China (973 Program, 2012CB720603), the
National Science Foundation of China (nos 21232005, 21472131,
J1310008 and J1103315), and the Specialized Research Fund for the
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