L. Lv, W. Luo and Q. Diao
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 246 (2021) 118959
Fig. 4. Frontier molecular orbital profiles of BPO-N3 and BPO-NH2 based on TDDFT (B3LYP/def2SVP-GD3(BJ)) calculations.
the probe can suitably be used in physiological environment. The exper-
iment on the effect of temperature (Fig. 3(B)) showed that the response
of the probe to H2S increased with the increase of temperature, and the
probe could effectively react with H2S at room temperature (25 °C).
Therefore, for convenience, we chose room temperature as the reaction
temperature in subsequent experiments. Fig. 3(C) shows the relation-
ship between the reaction time and the fluorescence intensity ratio,
which illustrates that the reaction reached the plateau after 30 min. Fi-
nally, we concluded that the optimal reaction conditions were pH 7.4,
room temperature and 30 min.
was obtained. The theoretical emission peaks of the two fluorescence
emissions were 555 and 624 nm, which were the results of the transi-
tion between the LUMO→HOMO energy levels. The electron cloud
caused by the transition from the azide group in the probe to amino
group was observed, and the push-pull electron effect in the molecule
was changed, which caused the red shift observed in the emission
spectrum.
3.6. Cell imaging
HepG2 cells were selected as the model cells. Prior to cell imaging,
MTT experiment was carried out to analyze the cytotoxicity of the
probe. The survival rate of the cells incubated with 50 μM BPO-N3 was
greater than 85%, indicating that the probe BPO-N3 has low cytotoxicity
(Fig. S7). We then carried out cell imaging experiment, and the results
are shown in Fig. 5. According to the results, HepG2 cells did not exhibit
fluorescence signal, but upon treating with the probe BPO-N3, obvious
green fluorescence signal was observed. After H2S was added to the
cells, the green fluorescence signal was significantly reduced, while
the red fluorescence signal was significantly enhanced. The ratio be-
tween the red and green fluorescence signals also showed that there
was a significant change of color, which indicates that H2S could instan-
taneously enter into the cell to react with the probe BPO-N3 and to gen-
erate the change of fluorescence signal. To further confirm that the
fluorescence change was caused by H2S, we pretreated HepG2 cells
with ZnCl2, which is an inhibitor of H2S [13], and the results showed
that there was no significant fluorescence change. Sodium nitroprusside
(SNP) was further used to stimulate the production of endogenous H2S
inside the cells [31]. We first incubated the cells with SNP for 30 min be-
fore adding BPO-N3. The imaging of the cells showed obvious red fluo-
rescence, indicating that the probe could also detect endogenous H2S.
Taken together, the probe BPO-N3 can sensitively monitor the concen-
tration of H2S in living cells.
3.4. Selectivity of probe
We investigated the effects of various interference species (includ-
ing inorganic salts, oxidizing species, proteins, and some sulfur-
containing compounds) on the probe BPO-N3. As illustrated in Fig. 3
(D), the fluorescence intensity ratio F625/F560 of the sample in the pres-
ence of H2S was significantly higher compared with that in the presence
of other species. This indicates that probe BPO-N3 is more selective to-
ward H2S than other species. Therefore, the probe BPO-N3 can suitably
be applied for the detection of H2S in biological systems.
3.5. Mechanism of reaction between probe and H2S
To study the reaction mechanism of BPO-N3 and H2S, the reaction
products were analyzed by ESI-MS. The mass spectrum data exhibited
a molecular ion peak at m/z = 263.0820, which is consistent with the
theoretical peak (m/z = 263.0815) of BPO-NH2 (the product of a reac-
tion between BPO-N3 and H2S) in CH3OH (Fig. S6).
In addition, we carried out TDDFT theoretical calculation to analyze
the optical properties of the probe BPO-N3 before and after the reaction
with H2S. Fig. 4 shows the structure of the corresponding ground and
excited states, and the electron cloud distribution in the corresponding
molecular orbital. The calculated maximum adsorption peak of BPO-N3
was 525 nm (f = 0.9466), and that of BPO-NH2 was 598 nm (f =
0.5013). The main electron transition occurred between the HOMO
and the LUMO energy levels, which is similar to the experimental re-
sults, in which the spectral red shift was observed. The structure of
the first excited state S0 was optimized, and the fluorescence emission
4. Conclusion
In this paper, we developed
a novel probe BPO-N3, using
phenoxazine as the fluorescence matrix, that has high sensitivity and
selectivity. The detection limit of the probe in the detection of H2S was
Fig. 5. Confocal fluorescence imaging the H2S in HepG-2 cells of BPO-N3. (A) Only HepG-2 cells (control).(B) Only incubated with BPO-N3 (10 μM) for 30 min. (C–E) Incubated with the
BPO-N3 (10 μM) for 30 min, and then incubated with 50, 100, 200 μM H2S for 30 min. (F) Incubated with the ZnCl2 (1 mM) for 1 h, and then incubated with BPO-N3 for 30 min, and then
incubated with 100 μM H2S for 30 min. (G) Incubated with the SNP (10 μM) for 1 h, and then incubated with BPO-N3 for 30 min. Excitation at 525 nm, the green channel was set at
550–580 nm, the red channel was set at 600–640 nm. Scale bar = 50 μm.
4