Sulfur dioxide (SO
2) is both a main atmospheric pollutant and a valuable commercial reagent, it is often produced through the combustion of sulfur-containing raw materials and fuels [
1]. It can dissolve in water and form a pH-dependent equilibrium between sulfite (SO
32−) and bisulfite (HSO
3−) with a 1:3 molar ratio [
2,
3]. In the industry, sulfur processing (including sulfur fumigation, pyrosulfite soaking,
etc.) is widely used to protect food, beverages, and Chinese medicinal materials from spoilage due to its antioxidant and enzyme inhibition properties [
4–
6]. For instance,
Pinelliae rhizoma (
P. rhizoma) is usually processed with fumigation or sodium pyrosulfite to ensure long-term storage and maintain good color [
7,
8]. However, a large amount of SO
2 usually remains in
P. rhizoma after treatment, which will reduce the medicinal ingredients of
P. rhizoma [
9], and the toxicological studies unveil that SO
2 is associated with lung cancer, cardiovascular diseases [
10], and neurological diseases [
11] may involve the formation of sulfur-and oxygen-centered free radicals that disturb the molecular mechanism of mitochondrial and affect cell physiological activity [
12]. Hence, the residual of SO
2 in Chinese medicinal materials is strictly controlled, and the amount of SO
2 should be <150 mg/kg according to Pharmacopoeia of People's Republic of China [
13]. Hence, developing a fast and efficient way for monitoring SO
2 residue in real Chinese herbal medicine is imperative.
Several methods for detecting SO
2 have been reported, including acid-base titration [
14], gas chromatography [
15], ion chromatography [
16], and fluorescence analysis [
17]. Among these methods, fluorescence analysis stands out for its simple operation, and high sensitivity [
18], especially the ratiometric and near-infrared (NIR) fluorescence, which has a lot of advantages: high accuracy, low autofluorescence interference, deep tissue penetration [
19,
20], rendering it ideal for quantitative detecting SO
2 residues in Chinese herbal medicine, and tracking effect of the SO
2 residues
in vivo [
21].
Herein, a mitochondrial-targeted fluorescent probe (
NIR-RS in
Scheme 1), that can be used for colorimetric and ratiometric detecting SO
2 in
P. rhizoma and
in vivo was developed. The coumarin-semirhodamine hybrid structure of probe
NIR-RS, features near-infrared fluorescence, excellent water solubility, and high quantum yield [
22,
23]. Upon addition of SO
2, probe
NIR-RS displays a distinct colorimetric and ratiometric fluorescence change in the far-red and NIR region (λ
em = 582 and 702 nm), and exhibited high sensitivity (Limit of detection (LOD) = 50 nmol/L), excellent selectivity. Importantly, probe
NIR-RS can accurately detect SO
2 content in
P. rhizoma samples, the recovery rates from 98.46% to 102.40%, with relative standard deviations (RSDs) < 5.0%. Moreover,
NIR-RS was successfully used to image both endogenous and exogenous SO
2 in mitochondria. Using
NIR-RS and
Rho-ATP (a probe for detecting adenosine triphosphate (ATP)) [
24] for optical imaging, we found that high SO
2 residue in
P. rhizoma increased ATP levels in mitochondria. This is the first fluorescent probe to evaluate the link between the high SO
2 residue of
P. rhizoma and ATP expression in cells. Additionally, when mice were orally administered
P. rhizoma with high SO
2 residue, SO
2 mainly accumulates in the liver, and can also cause pulmonary fibrosis and gastric mucosal damage. In summary, we developed a standardized tool for measuring SO
2 residue in
P. rhizoma and provided a new method for studying the metabolic effects of high SO
2 residue
in vivo.
First, compounds
1 and
2 were prepared by a literature method [
25,
26], and the synthesis of probe
NIR-RS is outlined in Scheme S2 (Supporting information). Compounds
1 and
2 in methylsulfonic acid at 120 ℃ to afford
NIR-RS in 23% yield. The structure of
NIR-RS was confirmed by nuclear magnetic resonance (NMR) and mass spectroscopy (Figs. S1–S4 in Supporting information).
Next, the performance of
NIR-RS for detecting SO
2 was studied in phosphate buffered solution (PBS) (10 mmol/L, pH 7.4, 30% dimethylformamide (DMF), v/v) by measuring the absorption and fluorescence changes. As expected,
NIR-RS has large conjugated structures and shows an absorption maxima at 660 nm and a fluorescence peak at 702 nm in the test solution (
Fig. 1). However, the maximum absorption peak of
NIR-RS at 660 nm was decreased abruptly when 200 µmol/L HSO
3− was added, and the absorption peak at 480 nm emerged, together with an obvious color change from green to pale pink (
Fig. 1a). This indicates that
NIR-RS can be used as a colorimetric probe for HSO
3−. Meanwhile, after HSO
3− was added, the fluorescence of
NIR-RS decreased significantly at 702 nm and concurrently increased at 582 nm (
Fig. 1b), the results indicate that
NIR-RS is a good ratiometric fluorescent probe for SO
32−. We also monitored the fluorescence intensity changes of
NIR-RS (10 µmol/L) at 582 and 702 nm after adding HSO
3−, HS
−, glutathione (GSH), homocysteine (Hcy), and cysteine (Cys), respectively, and kinetics results showed a fast reaction rate (<1 min, Fig. S6 in Supporting information) upon add SO
32−, and negligible changes were observed upon the addition of biothiol (HS
−, GSH, Hcy and Cys). Then, we investigated the response of
NIR-RS to various biologically relevant analytes, including Cl
−, I
−, HCO
3−, OCN
−, AcO
−, C
2O
42−, S
2O
72−, HSO
4−, F
−, Br
−, SCN
−, NO
2−, S
2O
32−, Ser, Glu, Lys, Arg, Gly, Phe, His, Ala, Met, Ile, Gln, Thr, Ca
2+, Zn
2+, Al
3+, Fe
3+, Cu
2+, Mg
2+), S
8, Na
2S
2, CH
3SSSCH
3. It was found only HSO
3− can cause a significant fluorescence change, this indicates that
NIR-RS is high selectivity for HSO
3− over other analytes (
Fig. 1c). Meanwhile, the competition experiments showed that all these biologically relevant analytes displayed almost no interference with HSO
3− detection (
Fig. 1d). What is more,
NIR-RS exhibited excellent photostability under 365 nm light exposure for 120 min (Fig. S7 in Supporting information). These illustrated that
NIR-RS displayed remarkable selectivity to HSO
3− detection. Then, we sought to investigate the pH effect on the fluorescence behavior of
NIR-RS in the detection of HSO
3−, As shown in Fig. S8 (Supporting information), the ratio of fluorescence intensities of
NIR-RS did not show significant changes, representing that this probe was stable in a wide range of pH. Notably, once HSO
3− was added to the
NIR-RS solution, the ratio of fluorescence intensities (
I582/
I702) was increased in the solution of
NIR-RS with SO
32− in the pH range of 6–11. This pH range for
NIR-RS in the detection of HSO
3− implied its potential application in environmental and biological studies.
After confirming the high selectivity of
NIR-RS for HSO
3−, then the sensitivity of
NIR-RS to SO
32− was investigated. As shown in
Fig. 2a, with increased concentrations of HSO
3−, the fluorescence of
NIR-RS decreased gradually at 702 nm while increasing obviously at 582 nm. The fluorescence intensities at 582 nm continued to rise and saturated after the addition of about 300 µmol/L of SO
2 derivative. A linear relationship (
R2 = 0.99333) was observed between the fluorescence intensity ratio (
I582/
I702) and HSO
3− concentration over the range of 0–200 µmol/L (
Fig. 2b). The detection limit was calculated to be 50 nmol/L, based on signal to noise ratio (S/N) = 3, indicating that
NIR-RS can be effectively employed for quantitative detection of HSO
3− over a broad linear range. These results suggest that
NIR-RS has a high sensitivity to HSO
3- and is suitable for detecting SO
2 derivatives in Chinese herbal medicine samples.
To investigate the application properties of
NIR-RS, we evaluated its effectiveness in determining SO
2 content in
P. rhizoma samples. First,
P. rhizoma samples treated with different concentrations of sodium pyrosulfite were measured by
NIR-RS. As shown in Table S1 (Supporting information), the probe successfully detected SO
2 content in the samples, with a relative standard deviation RSD of <5%. Furthermore, the results obtained from this method closely aligned with those from the national standard titration method (Table S2 in Supporting information) [
14].
T-test analysis revealed no statistically significant difference between the two methods (
P > 0.05), and the recovery rates of
NIR-RS detecting SO
2 in
P. rhizoma samples ranged from 98.46% to 102.40% (Table S3 in Supporting information). These results demonstrate that
NIR-RS is suitable for accurately and precisely detecting SO₂ content in
P. rhizoma samples. To our knowledge, few fluorescent probes are capable of detecting SO
2 in Chinese herbal medicine samples, which is likely attributable to the ratiometric property of
NIR-RS that enhances detection precision.
To understand the sensing mechanism of
NIR-RS for HSO
3−, considering the optical sign at 582 nm of
NIR-RS for detecting HSO
3− was increased, suggesting that
NIR-RSO showed the fluorescence of semi-rhodamine while
NIR-RS was taken addition with HSO
3− at the site 4 of coumarin, and the conjugated skeleton of
NIR-RS was interrupted and generated compound
NIR-RSO. To prove this, HPLC-MS analysis was carried out, and a mass peak at
m/
z 561.2391 corresponded to the addition adduct of N
IR-RS, in addition, the peak that appeared at
m/
z 641.1967 was attributed to compound
NIR-RSO (Fig. S9 in Supporting information). Thus, based on the reports of several recently reported semirhodamine-based HSO
3− probes [
27–
29], the sensing mechanism of probe
NIR-RS for HSO
3− was proposed as a process as shown in Scheme S2.
Before imaging, the MTT assays were used to evaluate the cytotoxicity of
NIR-RS, as shown in Fig. S10 (Supporting information),
NIR-RS exhibits no significant cytotoxicity. Mitochondria play essential roles in energy metabolism, and the SO
2 derivate was linked with mitochondrial disturbances [
30,
31]. The cationic probe
NIR-RS can easily bind to the mitochondrial membrane with a negative potential (−250 mV) [
32], which may accumulate in mitochondria. Then, the colocalization imaging experiment was performed to evaluate the distribution of
NIR-RS (Fig. S11 in Supporting information). When the
NIR-RS and a commercialized Mito-Tracker Green (
MTG) were co-incubated in cells the cytoplasm merged image showed the fluorescence distribution in green and red channels overlapped well, and the intensity profiles of the linear regions of interest (ROI) across the cells also displayed an excellent correlation. To further understand the distribution of the probe
NIR-RS in the cells, other commercial dyes, including Hoechst 33342 (nucleus), LysoTracker Green (lysosomes), GolgiTracker Green (Golgi), ER-Tracker Green (endoplasmic reticulum) was also incubated with probe
NIR-RS, as shown in Fig. S12 (Supporting information), we found that Pearson's co-localization was higher in mitochondria then other subcellular organelles. Additionally, the Pearson's co-localization and overlap coefficients in mitochondria were calculated to be 0.87 and 0.90, respectively. The above results indicated that
NIR-RS possesses a mitochondria-targeting capability with low cytotoxicity, and has the potential ability to track and image SO
2 in mitochondria.
Cell imaging experiments were carried out to study the imaging performance of the probe
NIR-RS for detecting SO
2. First, HeLa cells were incubated with
NIR-RS for 30 min, and strong red fluorescence and weak green fluorescence were observed, indicating that
NIR-RS has great cell membrane permeability (Fig. S13 in Supporting information). However, when the cells were incubated with different concentrations of exogenous SO
2 (HSO
3−), a remarkable increase in the green channel and a reduction in red fluorescence was observed (Figs. S13b and c). These results manifested that
NIR-RS could effectively detect exogenous SO
2. In addition, when cells were preincubated with Cys to produce endogenous SO
2 [
33], the fluorescence ratio of
Igreen/
Ired increased (Fig. S14 in Supporting information), and decreased in the presence of tetrahydrofolate (an endogenous formaldehyde (FA) reagent), this suggests that
NIR-RS has the potential to detect endogenous SO
2. Overall, these experiments show that
NIR-RS is suitable for imaging both exogenously and endogenously SO
2 in cells.
We further investigated the property of
NIR-RS to image SO
2 residual in
P. rhizoma sample within cells. As shown in
Fig. 3, when cells were incubated with
P. rhizoma sample containing different concentrations of SO
2, a dramatical increase in the green-to-red fluorescence ratio was observed, with a pronounced enhancement in the green channel and a decline in red channel fluorescence as SO
2 levels increased (
Fig. 3a). However, when FA was co-incubated with 5 g/kg
P. rhizoma sample, the red fluorescence was restored, and the green/red fluorescence ratio was depressed. These results indicated that
P. rhizoma fumigated with high sodium pyrosulfite content leads to elevated SO
2 expression in cells. Elevated SO
2 levels were found to increase the inner mitochondrial membrane potential (
Fig. 3b), which is reported to reflect an energized mitochondrion with enhanced ATP production capacity [
34]. Using
Rho-ATP (
Fig. 3c), we measured mitochondrial function by assessing ATP production (
Fig. 3d). Cells incubated with 5 mg/kg
P. rhizoma displayed an increase in green fluorescence, while cells treated with apyrase (a hydrolytic enzyme that can convert ATP into adenosine monophosphate (AMP) and inorganic phosphate) [
35–
37] showed weak green fluorescence. This indicates that SO
2-induced mitochondrial ATP production surged, potentially disturbing intracellular energy metabolism and impairing normal cellular function. Then, HeLa cells were pre-incubated with different concentrations of SO
2 residual from
P. rhizoma samples. Cells incubated with
P. rhizoma (1, 3, 5 g/kg) displayed an increase in green fluorescence compared to the blank group. The fluorescence intensity generally increased with higher SO
2 concentrations and reached a peak at 3 g/kg (Fig. S15 in Supporting information). These findings reveal that
P. rhizoma with excessive SO
2 can affect the organism's growth mechanisms through alterations in ATP pathways. This provides a new direction for researchers to explore the physiological effects of
P. rhizoma with excessive SO
2 residues
in vivo.Encouraged by the outcomes of the cellular experiments, we scrutinized the potential of NIR-RS to image SO2 in vivo. Kunming mice were purchased from the Hubei Provincial Center for Disease Control and Prevention (Wuhan, China). All the animal experiment procedures were approved by the Animal Experiment Center of Hubei University of Chinese Medicine and followed the Guidelines for the Care and Use of Research Animals. As shown in Fig. S16 (Supporting information), when the mouse was intravenously injected with NIR-RS, a strong fluorescence signal at 710 nm was observed. Upon orthotopic injection of SO32−, the 710 nm signal rapidly diminished, accompanied by a significant fluorescence increase at 570 nm within 1 min, indicating that NIR-RS can rapidly track SO32− in vivo. Then, we further investigated the property of NIR-RS to image SO2 residual from P. rhizoma sample in vivo. As shown in Fig. S17 (Supporting information), the 0 g/kg P. rhizoma group showed a stronger sign in the red channel. As the sodium pyrosulfite content in P. rhizoma increased, the fluorescence in the red channel decreased, while the green channel signal intensified. The results indicated that the NIR-RS could effectively detect SO2 residues in P. rhizoma samples in vivo.
To investigate the actual effects of excessive sulfur dioxide in the
P. rhizoma sample on organisms, Kunming mice were orally gavaged with 0.5 mL
P. rhizoma sample (0, 0.4, 5 g/kg) for 8 consecutive days. Histological analysis indicated the mice treated with
P. rhizoma sample (5 g/kg) exhibited signs of pulmonary fibrosis and gastric mucosal damage (Fig. S18 in Supporting information), while other tissues were minimally affected. In addition, tissue damage assessment experiments showed that the probe
NIR-RS was relatively safe at (100 µmol/L, in 100 µL PBS buffer solution) doses (Fig. S19 in Supporting information). Notably, as shown in
Figs. 4a–
d, SO
2 residues were detected
in vivo. In the 0 and 0.4 g/kg groups, distinct fluorescent signals were observed in the liver and kidneys at the 710 nm channel. Compared to the other two groups, the 0.4 g/kg group displayed significantly stronger fluorescence intensity (
Fig. 4f). This suggests that mice administered 0.4 g/kg
P. rhizoma sample may have reduced liver metabolic efficiency, leading to the accumulation of
NIR-RS in the liver. The ratiometric properties of the
NIR-RS probe indicated that SO
2 residue concentrations increased, and the SO
2 levels in the liver and kidneys also rose (
Figs. 4e and
f), indicating that SO
2 tends to accumulate in these organs. Additionally, the imaging confirmed the presence of excessive SO
2 residues in liver and kidney tissues (Fig. S20 in Supporting information), providing insights into the metabolic pathways for
P. rhizoma samples containing SO
2 residues.
In summary, we designed a fluorescent probe NIR-RS for detecting SO2 residues in P. rhizoma samples and living systems. Probe NIR-RS exhibits high sensitivity (LOD = 50 nmol/L), selectivity, and a rapid response for SO2 detection. It was successfully used for quantitative analysis of SO2 residues, with recovery rates ranging from 98.46% to 102.40% and RSDs < 5.0%. In addition, NIR-RS has low cytotoxicity and good mitochondrial-targeting ability to image both mitochondrial exogenous and endogenous SO2. Importantly, we found that P. rhizoma with high SO2 residues increases ATP levels in mitochondria, Probe NIR-RS imaging in vivo demonstrated its excellent ability to monitor SO2 residue metabolism, and excessive intake of P. rhizoma containing high SO2 residue can cause pulmonary fibrosis and gastric mucosal damage. Overall, this study provides a powerful tool for regulating SO2 residue levels in P. rhizoma and offers new methods for tracking SO2 metabolism in vivo. It also opens new avenues for researching the pathological and physiological toxicity of SO2 residues in P. rhizoma.
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