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• Sulfydryl-contained (-SH) substances, namely biothiols, including hydrogen sulfide (H₂S), cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) are important reductants in living systems, which are crucial in maintaining redox homeostasis and participating in signal transduction [1–5]. H₂S is the third vital signaling molecule after carbon monoxide (CO) and nitric oxide (NO) in living system, and it is produced from Cys and Hcy via enzymatic metabolism of cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST) [6–9]. In signaling processes, there is a rapid fluctuation in the concentration of endogenous H₂S from nanomolar to sub millimolar levels [10]. Generally, the abnormal variation of H₂S is associated with acute liver/lung injury [11,12], nonalcoholic fatty liver [13,14], Alzheimer's disease [15], Parkinson's disease [16], inflammation [17], cancer [18,19], etc. Cys/Hcy/GSH are considered the most abundant biothiols, which are found in the concentration ranges of 240–360 µmol/L (Cys), 12–15 µmol/L (Hcy), and 1–10 mmol/L (GSH) in living beings [20]. The abnormal fluctuations of Cys/Hcy/GSH are associated with liver damage [21,22], muscle loss [23], diabetes [24], atherosclerotic cardiovascular disease [25], cancer [26,27], epilepsy [28], etc. As depicted in Fig. 1b, H₂S and Cys/Hcy/GSH formed a transformation network in organism, which highlights the importance of distinguishing H₂S and Cys/Hcy/GSH to understand their transformation network, metabolic process and the related pathological characteristics.

  Figure 1

  

  Figure 1.  (a) Mechanism of Y-NBD responding to Cys/Hcy and H₂S in this work. (b) Schematic diagram of the mutual conversion of Hcy/Cys and the generation of H₂S. The abbreviations in Fig. 1b are presented in Supporting information.

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  The fluorescent probe, especially the activatable near-infrared (NIR) fluorescent probe, has attracted tremendous concerns due to the merits of high sensitivity, low background signal, real-time and in-situ imaging [29–36]. In the past decades, a large number of Cys/Hcy/GSH and H₂S specific fluorescent probes have been reported based on the mechanism of nucleophilic aromatic substitution reaction (S_NAr) [37–39]. However, owing to their similar reactivity and rapid transformation in living systems, dynamic differentiating biothiols by one probe is still challenging. In fact, -SH-contained substances have different pK_a values (6.9 (H₂S), 8.3 (Cys), 8.9 (Hcy), and 9.2 (GSH)) [40], which suggests that H₂S has a stronger nucleophilicity than other biothiols. Based on this, some fluorescent probes were reported for selective detection of single thiol [39], or simultaneous detection of Cys/Hcy/GSH/H₂S with multiple fluorescent signals [41,42]. Nevertheless, the NIR fluorescence probe that simultaneously responds to Cys/Hcy and H₂S with high signal-background-ratio (SBR) without recognizing GSH has rarely been reported.

  In this work, we constructed an intramolecular charge transfer (ICT)-based NIR fluorescent probe Y-NBD by using dicyanoisophorone-coumarin as fluorophore and 7-nitro-1,2,3-benzoxadiazole (NBD) as recognition unit. Y-NBD exhibits high selectivity towards Cys/Hcy and H₂S (Fig. 1a), showing excellent capability of imaging the exogenous/endogenous thiols in various living cells and zebrafish. Furthermore, Y-NBD shows a certain potency to investigate variations of Cys/Hcy and H₂S in drug-induced liver injury and its remediation.

  To design a NIR fluorescent probe for the differentiation of several biothiols with high SBR, the ICT mechanism was chosen to regulate the fluorescence. First, the dicyanoisophorone [43] was selected to bridge the aldehyde coumarin derivative to enlarge the conjugation system forming the fluorophore Y-OH, which would be an ICT-controllable NIR fluorophore with a large Stokes shift. NBD chromophore, as recognition unit and fluorescence masking unit, was linked to fluorophore Y-OH a by an ether bond to obtain probe Y-NBD (Fig. S1 in Supporting information), and its characteristic data were presented in Supporting information.

  With the targeted probe Y-NBD in hand, its recognition selectivity was first examined by absorption and fluorescence spectra. The absorption spectra of Y-NBD displayed significant changes upon addition of Cys/Hcy and H₂S. As shown in Fig. 2a, the Y-NBD shows a ~55 nm redshift for Cys/Hcy (100 µmol/L) and a ~140 nm redshift for H₂S (100 µmol/L). Moreover, free Y-NBD shows almost no background fluorescence, whereas Cys/Hcy induces remarkable fluorescence signals at 555 nm (Fig. 2b) and H₂S at 719 nm (Fig. 2c) with 30-fold/242-fold and 27-fold enhancement, respectively. Notably, the absorption and fluorescence spectra of Y-NBD exhibit negligible changes upon addition of GSH, even the concentration of GSH up to 5 mmol/L (Fig. S2 in Supporting information), indicating that Y-NBD can differentiate Cys/Hcy and H₂S with high SBR and indeed cannot respond to GSH.

  Figure 2

  

  Figure 2.  Ultraviolet absorbance (a) and fluorescence spectra (b, λ_ex = 470 nm; c, λ_ex = 550 nm) of Y-NBD (10 µmol/L) upon addition of Cys/Hcy/H₂S (100 µmol/L) in CH₃CN/PBS (4:6, v/v). (d–f) Fluorescence spectra of Y-NBD (10 µmol/L) in presence of various concentrations of Cys/Hcy/H₂S, the insert is the corresponding fluorescent intensity and the concentration of the analyte. (g–i) Time-dependent fluorescence intensity of Y-NBD (10 µmol/L) in absence and presence of Cys (100 µmol/L) (g), Hcy (100 µmol/L) (h) at 555 nm and H₂S (100 µmol/L) (i) at 719 nm. (j–l) Stokes shift of Y-NBD in presence of Cys (j), Hcy (k), and H₂S (l).

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  Subsequently, titration experiments were carried out to check the quantitative capability of Y-NBD for Cys/Hcy and H₂S. Absorption of Y-NBD exhibited redshift and ratio changes upon addition of various concentrations of Cys/Hcy and H₂S (Figs. S3a–c in Supporting information). Meanwhile, the fluorescence intensity of Y-NBD solution at both 555 nm and 719 nm were gradually increased upon addition of Cys/Hcy (0–500 µmol/L) (Figs. 2d and e, Figs. S4a and b in Supporting information) with a linear fit at 0–50 µmol/L for Cys (R² = 0.9909) and 0–20 µmol/L for Hcy (R² = 0.9948) (Figs. S5a and b in Supporting information). Simultaneously, as the concentration of H₂S gradually increased (0–500 µmol/L), the fluorescent intensity of Y-NBD solution was significantly increased merely at 719 nm with a linear relationship at 0–100 µmol/L (R² = 0.9903) (Fig. 2f and Fig. S5c in Supporting information). The limits of detection (LOD = 3σ/K) were determined to be 79 nmol/L (Cys), 24 nmol/L (Hcy) and 203 nmol/L (H₂S), suggesting Y-NBD is suitable for quantitative detection of low-concentration thiols.

  To evaluate the application capability of Y-NBD in complex living systems, the anti-interference of Y-NBD towards Cys/Hcy and H₂S in presence of other analytes were investigated, indicating that almost no significant fluorescence changes were observed when Cys/Hcy and H₂S coexisted with various species except for HClO, which showed a slight decrease in fluorescence intensity at 555 nm (Figs. S5d–f in Supporting information). It would be ascribed to the oxidizability of HClO causing part inactivation of Cys/Hcy. Considering a relatively low concentration of HClO in the biological system, its interference could be neglected. The effect of pH showed that free Y-NBD possesses good pH stability at a wide pH range, while the fluorescence intensity of Y-NBD in presence of H₂S showed dramatically increase under alkaline conditions due to the generated compound Y-OH existed in “basic form” (pK_a = 7.62) (Figs. S6 and S7 in Supporting information). Moreover, time-dependent fluorescence spectra showed that Cys-induced fluorescence increased within 20 min and reached a plateau at 40 min (Fig. 2g), while Hcy was slower than Cys, which increased within 30 min (Fig. 2h). By comparison, H₂S-induced fluorescence intensity increased quickly within 10 min and reached a plateau at 20 min (Fig. 2i), which is attributed to its higher nucleophilicity than Cys/Hcy. More importantly, the Stokes shift of Y-NBD for Cys/Hcy and H₂S are 100/105 nm and 191 nm, respectively (Figs. 2j–l). Such a large Stokes shift would effectively avoid false positive signals resulting from excitation light in the application of confocal imaging.

  The recognition mechanism was explored and proposed according to the reported work [44–47]. As depicted in Fig. S8a (Supporting information), the -SH group of H₂S/Cys/Hcy reacted with Y-NBD via S_NAr reaction along with the release of fluorophore Y-OH. The intermediate of five/six member-rings formed as a result of recombination between the -NH₂ group and the -SH group of Cys/Hcy. All the reaction products were consistent with the corresponding HRMS data (Fig. S9 in Supporting information), indicating the feasibility of the deduced recognition mechanism. To further explore whether the fluorescent signal was regulated by the ICT process, theoretical calculation was carried out. As illustrated in Fig. S8b (Supporting information), the electrostatic potential (ESP) of Y-OH exhibited negative-to-positive (negative in red and positive in blue), which would be well matched with ICT process with dramatical fluorescence, while the ESP of Y-NBD showed negative-to-negative, resulting in the break of ICT process with none-fluorescence. In addition, the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) distributions were calculated. Compared with the compound Y-OH, the HOMO distribution of Y-NBD only was in the plane of the fluorophore, while the LUMO distribution of Y-NBD was in both recognition unit and fluorophore (Fig. S8c in Supporting information), suggesting that Y-NBD would afford a moderate reactivity for nucleophile species. This is also different from the GSH-specific fluorescent probe that LUMO distribution locates only in the recognition unit [21,48], which properly supported that Y-NBD only responds to Cys/Hcy and H₂S, but cannot react with GSH.

  To check the capability of Y-NBD to image Cys/Hcy and H₂S in living systems, the cytotoxicity of the Y-NBD was first evaluated by MTT assay. As shown in Fig. S10 (Supporting information), the cell viability of living cells (HeLa, A549, and HepG2) were more than 80% after incubation of Y-NBD (10 µmol/L) for 24 h, suggesting low toxicity of Y-NBD. Subsequently, Y-NBD was utilized to image intracellular Cys/Hcy and H₂S. As shown in Fig. S11a (Supporting information), both the green channel and red channel showed significant fluorescence signals in living HeLa, A549, and HepG2 cells. Moreover, it seems that the fluorescent signal in HepG2 cells was higher than that in A549 cells and HeLa cells (Fig. S11b in Supporting information), revealing that HepG2 cells may contain more Cys/Hcy and H₂S than A549 and HeLa cells. Thus, the HepG2 cell was chosen to perform the subsequent experiments.

  The distribution and transformation of Cys/Hcy and H₂S in living cells were further investigated. Compared with the cells only incubated with Y-NBD (Fig. 3a1), the cells pretreated with N-ethylmaleimide (NEM, a thiol scavenger) and then incubated with Y-NBD exhibited barely fluorescence signals in both green channel and red channel (Fig. 3a2). Meanwhile, when cells were pretreated with NEM followed by upon addition of Cys/Hcy or H₂S, the fluorescence recovered obviously (Figs. 3a3–a5 and b), indicating that Y-NBD could be utilized to monitor exogenous Cys/Hcy and H₂S. In addition, when N-acetylcysteine (NAC, a precursor of cysteine) was added to cells before incubation of Y-NBD, the fluorescence signals were dramatically increased in both green channel and red channel (Fig. 3a6). Afterward, we explored Y-NBD to image endogenous H₂S. Given that CSE is one of the important enzymes to catalyze Cys convert to H₂S [9,49], and dl-propargylglycine (PAG) is a famous inhibitor of CSE [50,51]. We thus used PAG to inhibit CSE to transform Cys into H₂S as a control, which observed a significant decrease of fluorescence in red channel (Fig. 3a7). Moreover, by using GSH to elevate the endogenous Cys and induce H₂S production [52], the fluorescence in the two channels were increased compared with the control group (Figs. 3a8 and b), suggesting it is reasonable to convert Cys or GSH into intracellular H₂S. 3D imaging showed that the fluorescence signals were located intracellular with high SBR (Fig. 3c). The results manifested Y-NBD could image endogenous Cys/Hcy and H₂S and illustrate the transformation relationship between several intracellular thiols with excellent imaging performance.

  Figure 3

  

  Figure 3.  (a) Confocal images of the detection Cys/Hcy/H₂S in HepG2 cells (Scale bar: 50 µm). (a1) Cells incubated with Y-NBD; (a2–a5) cells pretreated with NEM (100 µmol/L, 30 min) followed by incubation with Cys (a3), Hcy (a4), and H₂S (a5) (50 µmol/L, 30 min) and then upon addition of Y-NBD; (a6–a8) cells pretreated with NAC (100 µmol/L, 6 h) (a6), PAG (200 µmol/L, 6 h) (a7), GSH (200 µmol/L, 6 h) (a8), and then incubated with Y-NBD. (b) Relative fluorescence intensity of Fig. 3a conducted by the software of Image J. (c) Z-axis scanning and three-dimensional reconstruction in green and red channels in living HepG2 cells. (d) Confocal imaging of zebrafish (Scale bar: 1 mm). (d1) Zebrafish incubated with Y-NBD (10 µmol/L) for 30 min; (d2) zebrafish treated with NEM (1 mmol/L, 30 min) and then upon addition of Y-NBD; (d3–d5) zebrafish pretreated with NEM (1 mmol/L, 30 min) followed by incubation with Cys/Hcy/H₂S (50 µmol/L, 30 min) and then upon addition of Y-NBD. (e) Enlargement of merge and overlay in Fig. 3d3.

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  Considering zebrafish and their embryos hold significant potential for advancing research on human diseases because zebrafish possess a high level of genetic homology (approximately 87%) with humans [53,54], the imaging capability of the Y-NBD in zebrafish was examined. All the animal experiments have been approved by the Animal Ethics Committee of Northwest University (NWU-AWC-20230403M). As shown in Fig. 3d1, Y-NBD exhibited obvious signals in both green and red channels in zebrafish. However, with the pretreatment of NEM, there was almost no fluorescent signal was observed (Fig. 3d2). Meanwhile, when exogenous Cys/Hcy or H₂S were added, the fluorescent signals were observed obviously (Fig. 3d3–d5) and mainly existed in the intestine (Fig. 3e). It manifests that Y-NBD exerts well imaging ability for detecting Cys/Hcy and H₂S in zebrafish, and Y-NBD would be metabolized through the intestine of zebrafish. Furthermore, given that the concentration of thiols decreases dramatically during drug-induced liver injury (DILI) [11,55–57], we further examined the capability of Y-NBD to evaluate DILI. As illustrated in Figs. 4a and b, when cells were pretreated with overdose acetaminophen (APAP, a highly utilized medication for relief of pain and fever but overdose leads to liver injury [58]) followed by the incubation of Y-NBD, the fluorescence signals decreased in two channels. However, after treatment of NAC or GSH, the fluorescence showed a significant enhancement in both green and red channels, suggesting that both NAC and GSH have certain efficacy in alleviating liver injury. Besides, the fluorescence intensity of the cells treated with NAC was higher than that treated with GSH, indicating that NAC would be a more efficient drug to treat APAP-induced liver injury.

  Figure 4

  

  Figure 4.  (a) Fluorescence imaging of APAP-treated cells and remediation (Scale bar: 50 µm). (a1) Cells are incubated with Y-NBD; (a2–a4) Cells pretreated with APAP (5 mmol/L) and then upon addition of NAC (1 mmol/L) (a3) or GSH (1 mmol/L) (a4) before incubation of Y-NBD. (b) Relative fluorescence intensity of Fig. 4a conducted by the software of Image J. (c) APAP-induced injury liver imaging and HE/Masson staining. (c1) Control group, the liver from normal mouse with intraperitoneal injection of Y-NBD. (c2) DILI group, the liver from the mouse pretreated with overdose APAP (350 mg/kg) before intraperitoneal injection of Y-NBD. (c3) Remediation group, the liver from the mouse with intraperitoneal injection of NAC (200 mg kg⁻¹ d⁻¹) for 5 days, and then intraperitoneal injection of Y-NBD (1 mmol/L, 500 µL) 1 h after gavage APAP (350 mg/kg), (yellow arrow: inflammatory cell infiltration; blue arrow: fibrosis in tissues; scale bar: 50 µm). (d) Fluorescence semi-quantitative analysis of Fig. 4c.

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  To check the DILI imaging capability of Y-NBD in liver tissue, the biosafety of Y-NBD was evaluated first by hematoxylin-eosin (HE) staining. As depicted in Fig. S12 (Supporting information), after administration of Y-NBD, no obvious histological morphology changes of main organs (heart, liver, spleen, lung, kidney) were observed compared with the control group, suggesting that the Y-NBD has good biosafety in vivo. Then the APAP-induced liver injury mice model was constructed to evaluate DILI by using Y-NBD. As shown in Fig. 4c2, the liver from the mouse pretreated with overdose-APAP shows a remarkable decrease in fluorescence signal compared with the control group (Fig. 4c1), while a recovered fluorescence signal was observed after being treated with NAC (Fig. 4c3), indicating that Y-NBD has a tremendous potency to visualize liver injury and its remediation. Besides, HE and staining and Masson's trichrome (Masson) staining were performed. As shown in Fig. 4c5, the overdose-APAP treated group shows a local aggregation of inflammatory cell infiltration compared with the control group (Fig. 4c4), while after treatment with NAC, this phenomenon reduced obviously (Fig. 4c6). Masson staining shows that the overdose-APAP treated group shows mild fibrosis in the hepatocellular stroma (Fig. 4c8) compared with the control group (Fig. 4c7) and the degree of fibrosis decreased after treatment of NAC (Fig. 4c9). These staining results of HE and Masson further validate the feasibility and accuracy of Y-NBD for assessing the degree of DILI and its treatment efficiency of drugs.

  In summary, we developed an ICT-based NIR fluorescent probe Y-NBD by using dicyanoisophorone-coumarin as fluorophore and nitrobenzoxazole as the recognition unit. Y-NBD enables differentiation of Cys/Hcy and H₂S (not responding to GSH) with high SBR and large Stokes shift. It can be applied to visualize exogenous and endogenous Cys/Hcy and H₂S in living cells and zebrafish. The results suggest that HepG2 cells contain more Cys/Hcy and H₂S than other cell lines, and Y-NBD is mainly activated and metabolized in the intestine of zebrafish. Moreover, Y-NBD was applied to monitor the conversion of Cys/Hcy or GSH into H₂S in living HepG2 cells, as well as assess APAP-induced liver injury and its remediation. This work not only provides a valuable fluorescent probe Y-NBD to visualize the transformation pathway of Cys/Hcy and H₂S in living systems but also affords a promising tool for evaluating hepatotoxicity and its treatment efficiency in drug discovery.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  Acknowledgments

  This work was supported by the National Natural Science Foundation of China (Nos. 22077099 and 22171223), the Innovation Capability Support Program of Shaanxi (Nos. 2023-CX-TD-75 and 2022KJXX-32), the Technology Innovation Leading Program of Shaanxi (Program No. 2023KXJ-209), the Natural Science Basic Research Program of Shaanxi (Nos. 2022JQ-151 and 2023-JC-YB-141), and Young Talent Fund of Association for Science and Technology in Shaanxi, China (No. SWYY202206), the Shaanxi Fundamental Science Research Project for Chemistry & Biology (Nos. 22JHZ010 and 22JHQ080), the Yan'an City Science and Technology Project (No. 2022SLZDCY-002).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2023.109156.

  
  
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  Figure 1  (a) Mechanism of Y-NBD responding to Cys/Hcy and H₂S in this work. (b) Schematic diagram of the mutual conversion of Hcy/Cys and the generation of H₂S. The abbreviations in Fig. 1b are presented in Supporting information.

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  Figure 2  Ultraviolet absorbance (a) and fluorescence spectra (b, λ_ex = 470 nm; c, λ_ex = 550 nm) of Y-NBD (10 µmol/L) upon addition of Cys/Hcy/H₂S (100 µmol/L) in CH₃CN/PBS (4:6, v/v). (d–f) Fluorescence spectra of Y-NBD (10 µmol/L) in presence of various concentrations of Cys/Hcy/H₂S, the insert is the corresponding fluorescent intensity and the concentration of the analyte. (g–i) Time-dependent fluorescence intensity of Y-NBD (10 µmol/L) in absence and presence of Cys (100 µmol/L) (g), Hcy (100 µmol/L) (h) at 555 nm and H₂S (100 µmol/L) (i) at 719 nm. (j–l) Stokes shift of Y-NBD in presence of Cys (j), Hcy (k), and H₂S (l).

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  Figure 3  (a) Confocal images of the detection Cys/Hcy/H₂S in HepG2 cells (Scale bar: 50 µm). (a1) Cells incubated with Y-NBD; (a2–a5) cells pretreated with NEM (100 µmol/L, 30 min) followed by incubation with Cys (a3), Hcy (a4), and H₂S (a5) (50 µmol/L, 30 min) and then upon addition of Y-NBD; (a6–a8) cells pretreated with NAC (100 µmol/L, 6 h) (a6), PAG (200 µmol/L, 6 h) (a7), GSH (200 µmol/L, 6 h) (a8), and then incubated with Y-NBD. (b) Relative fluorescence intensity of Fig. 3a conducted by the software of Image J. (c) Z-axis scanning and three-dimensional reconstruction in green and red channels in living HepG2 cells. (d) Confocal imaging of zebrafish (Scale bar: 1 mm). (d1) Zebrafish incubated with Y-NBD (10 µmol/L) for 30 min; (d2) zebrafish treated with NEM (1 mmol/L, 30 min) and then upon addition of Y-NBD; (d3–d5) zebrafish pretreated with NEM (1 mmol/L, 30 min) followed by incubation with Cys/Hcy/H₂S (50 µmol/L, 30 min) and then upon addition of Y-NBD. (e) Enlargement of merge and overlay in Fig. 3d3.

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  Figure 4  (a) Fluorescence imaging of APAP-treated cells and remediation (Scale bar: 50 µm). (a1) Cells are incubated with Y-NBD; (a2–a4) Cells pretreated with APAP (5 mmol/L) and then upon addition of NAC (1 mmol/L) (a3) or GSH (1 mmol/L) (a4) before incubation of Y-NBD. (b) Relative fluorescence intensity of Fig. 4a conducted by the software of Image J. (c) APAP-induced injury liver imaging and HE/Masson staining. (c1) Control group, the liver from normal mouse with intraperitoneal injection of Y-NBD. (c2) DILI group, the liver from the mouse pretreated with overdose APAP (350 mg/kg) before intraperitoneal injection of Y-NBD. (c3) Remediation group, the liver from the mouse with intraperitoneal injection of NAC (200 mg kg⁻¹ d⁻¹) for 5 days, and then intraperitoneal injection of Y-NBD (1 mmol/L, 500 µL) 1 h after gavage APAP (350 mg/kg), (yellow arrow: inflammatory cell infiltration; blue arrow: fibrosis in tissues; scale bar: 50 µm). (d) Fluorescence semi-quantitative analysis of Fig. 4c.

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