All reagents were obtained from commercial suppliers and were used and stored according to the manufacturer’s specifications. Specifically, 5-Bromothiophene-2-Carbaldehyde (Catalog No. B801990), 4-Hydroxyphenylboronic Acid (Catalog No. H804864), Tetrakis (triphenylphosphine) palladium (Catalog No. T819527), 2,4-Dinitrobenzenesulfonyl Chloride (Catalog No. D830587), dimethyl sulfoxide (Catalog No. D806645), and deuterated dimethyl sulfoxide (Catalog No. D807976) were sourced from Shanghai Maclin Biochemical Technology Co., Ltd. (Shanghai, China). potassium carbonate (Catalog No. A17026), tetrahydrofuran (Catalog No. W310137), methanol (Catalog No. A04002925) were sourced from Shanghai Titan Scientific Technology Co., Ltd. (Shanghai, China). Dulbecco’s Modified Eagle’s Medium (Catalog No. KGL1202-500) and phosphate-buffered saline (Catalog No. KGL2206-500) were obtained from Jiangsu Kaiji Biotechnology Co., Ltd. (Jiangsu, China). Additionally, Thiazoyl blue tetrazolium bromide (MTT) (Catalog No: L11939.03) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Nuclear magnetic resonance (NMR) spectroscopy was performed using a Bruker 600 MHz NMR spectrometer (AVANCE NEO 600MHz, Bruker Corporation, Fällanden, Switzerland). LC-MS analyses were conducted on an ISQEM-ESI system from Thermo Fisher Scientific. UV-Vis absorption spectra were recorded using an Agilent Cary 60 spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA), while fluorescence spectra were measured with a Lumina fluorescence spectrophotometer from Thermo Fisher Scientific. Cellular fluorescence imaging was conducted using a Carl Zeiss LSM800 confocal laser scanning microscope (Carl Zeiss AG, Oberkochen, German).

The synthetic pathway for the probe DCMOS-N is illustrated in Fig. 2, with detailed synthetic procedures provided in the synthesis section of the Supporting Information. The chemical structures of all intermediates and the final probe were thoroughly characterized using ¹H NMR, ¹³C NMR, and LC-MS (Supplementary Material-Supporting Information, Supplementary Figs. 1–12).

Fig. 2.

Synthetic route for DCMOS-N.

To overcome the challenges of short emission wavelengths and limited Stokes shifts in probe molecules, dicyanomethylene-4H-pyran (DCM) was chosen as the fluorophore due to its excellent photochemical and chemical stability, as well as its tunable emission properties. DCM was modified through a Knoevenagel condensation reaction, extending its conjugated system and resulting in the formation of DCMOS, which shifted the fluorescence emission into the near-infrared region (with a maximum emission wavelength of 651 nm). Additionally, DNBS was introduced as a recognition unit for biothiols, yielding the NIR fluorescent probe by linking the fluorophore DCMOS to the strong electron-withdrawing DNBS group via a sulfonic ester linker.

The general method for spectroscopic analysis is outlined below unless stated otherwise. Stock solutions of the DCMOS fluorophore and probe DCMOS-N were prepared at a concentration of 10 mM in DMSO. For testing, a THF/PBS buffer solution was used. The spectroscopic properties of DCMOS-N were analyzed at a concentration of 10 µmol/L. The interaction between probe DCMOS-N and biothiols was evaluated in a THF/PBS buffer mixture (v/v = 4/6, pH = 7.4). The mixture was then transferred to a quartz cuvette with a 1 cm path length for fluorescence measurements, using an excitation wavelength of 480 nm and an emission wavelength of 651 nm, with both slit widths set to 10 nm.

HeLa cells (CL-0101) were kindly provided by Wuhan Pricella Biotechnology Co., Ltd. (Wuhan, China) and cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, maintained in a CO₂ incubator at 37 °C with 5% CO₂ and 95% air. The cell line was validated by STR profiling and tested negative for mycoplasma. The cytotoxicity of DCMOS and DCMOS-N was evaluated using the Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. A sterilized 96-well plate was used, with each well containing 100 µL of HeLa cell suspension at a density of 5 $\times$ 10³ cells per well. The cells were allowed to adhere for 24 hours before being treated with DCMOS and DCMOS-N at concentrations of 1, 2.5, 5, 7.5, 10, 15, 20, and 30 µmol/L. They were then incubated at 37 °C in 5% CO₂ for an additional 24 hours. Following incubation, the medium was removed, and 100 µL of DMSO was added to each well to dissolve the formazan crystals with shaking. Absorbance was measured at 490 nm using a Thermo Fisher Varioskan Flash multimode microplate reader (Thermo Fisher Scientific). Cell viability at 0 µmol/L probe concentration was set to 100%. The percentage of cell viability was calculated using the following formula: Cell viability (%) = (Mean optical density of the sample $\times$ 100)/(Mean optical density of the control group).

Cell imaging at varying concentration gradients was performed by treating HeLa cells with 20 µmol/L DCMOS-N. Fluorescence images were acquired using a Carl Zeiss LSM800 confocal laser scanning microscope. Imaging parameters included a $\times$63 objective, 12% laser intensity, a 40 µm pinhole size, and a voltage gain of 750 V. The excitation wavelength was set at 488 nm, with emission captured in the 600–700 nm range.

To evaluate the effectiveness of DCMOS-N, preliminary experiments were conducted to determine the optimal conditions for detecting biothiols (GSH, Cys, Hcy). THF/PBS (4/6, v/v, 10 µmol/L, pH 7.4) was identified as the most suitable testing environment (Supplementary Figs. 13,14). Subsequently, the absorption and fluorescence spectra of probe DCMOS-N (10 µmol/L) were recorded before and after interaction with biothiols (500 µmol/L). As illustrated in Fig. 3a, he unreacted probe DCMOS-N exhibited a weak absorption peak at 470 nm, which disappeared upon the addition of biothiols, while a new peak emerged at 493 nm, closely matching the absorption peak of DCMOS. Fig. 3b illustrates that initially, probe DCMOS-N displayed minimal fluorescence. Notably, a significant fluorescence enhancement at 651 nm was observed upon the addition of biothiols, indicating a structural change in DCMOS-N upon reaction with biothiols. Furthermore, kinetic studies of DCMOS-N with GSH, Cys, and Hcy at 651 nm revealed peak fluorescence intensities at approximately 2, 5, and 7 minutes, with significant enhancements of 10-, 7-, and 14.5-fold, respectively Fig. 3c. Additionally, the interaction product DCMOS, formed from the reaction between DCMOS-N and biothiols, exhibited a substantial Stokes shift of 151 nm, making it highly advantageous for bioimaging applications (Supplementary Fig. 15).

Fig. 3.

Investigation of the characteristics of the probe DCMOS-N. Absorption (a) and fluorescence spectra (b) of probe DCMOS-N (10 µmol/L) with biothiols in THF/PBS (4/6, v/v, 10 µmol/L, pH 7.4). (c) The reaction kinetics of DCMOS-N with biothiols. (d–f) The fluorescence selectivity and competitive analysis of DCMOS-N (10 µmol/L) upon the addition of various analytes (Fe³⁺, Na⁺, Zn²⁺, Mg²⁺, K⁺, H₂O₂, S^2-, HSO₃^–, SO₃^2-, and SO₄^2-, glycine (Gly), leucine (Leu), arginine (Arg), tyrosine (Tyr), glutamic acid (Glu), threonine (Thr), tryptophan (Trp), GSH, Cys and Hcy each at 500 µmol/L). ($\lambda$ex = 480 nm). FL, fluorescence.

Selectivity and anti-interference capabilities are crucial for evaluating the effectiveness of a fluorescent probe. To assess these properties, DCMOS-N was tested against various ions, including Fe³⁺, Na⁺, Zn²⁺, Mg²⁺, K⁺, H₂O₂, S^2-, HSO₃^–, SO₃^2-, and SO₄^2-, as well as amino acids such as glycine (Gly), leucine (Leu), arginine (Arg), tyrosine (Tyr), glutamic acid (Glu), threonine (Thr), tryptophan (Trp), alongside biothiols (Cys, Hcy, and GSH). As illustrated in Fig. 3d–f, a significant fluorescence enhancement was observed only in the biothiol test groups, indicating strong selectivity. Additionally, the anti-interference performance of DCMOS-N was evaluated, demonstrating that the presence of other potential interferents had no impact on its ability to detect biothiols. These findings confirm that DCMOS-N exhibits high selectivity and strong anti-interference properties, highlighting its potential for biothiol detection in live-cell bioimaging.

To thoroughly investigate the interaction between DCMOS-N and biothiols, a concentration titration experiment was conducted with biothiol concentrations ranging from 0 to 1 mmol/L (Fig. 4). First, DCMOS-N exhibited negligible fluorescence emission; however, a gradual increase in fluorescence intensity was observed as the biothiol concentration increased (Fig. 4a–c). The fluorescence intensity at 651 nm was measured and recorded, and the resulting data were used to generate response curves (Fig. 4a–c, inset). A strong linear correlation was observed within the concentration range of 0 to 100 µmol/L, with R² values of 0.9944 for GSH, 0.9942 for Cys, and 0.9946 for Hcy (Fig. 4d–f, inset), enabling the quantitative analysis of biothiol concentrations in biological systems. Additionally, the limits of detection (LOD) for GSH, Cys, and Hcy were calculated to be 0.142, 0.129, and 0.143 µmol/L, respectively (Supplementary Figs. 16–18). Moreover, a comparative analysis of recently developed fluorescent probes employing 2,4-dinitrobenzenesulfonyl (DNBS) as a recognition moiety, including LOD, media, and applications, is presented in Supplementary Table 1. The probe DCMOS-N has advantages in terms of LOD, with higher sensitivity. These findings confirm that DCMOS-N can sensitively detect biothiols, facilitating fluorescence-based detection even at low concentrations.

Fig. 4.

Fluorescence spectra of probe DCMOS-N in response to biothiols of different concentrations. Fluorescence spectra of probe DCMOS-N (10 µmol/L) in response to three biothiols: GSH (a), Cys (b), and Hcy (c) at varying concentrations (0–1 mmol/L). Insets in (a–c): Fluorescence intensity of probe DCMOS-N (10 µmol/L) at different biothiol concentrations (0–1 mmol/L). Insets in (d–f): Linear relationship between the fluorescence intensity ratio (F/F₀) and biothiol concentration (0–100 µmol/L). ($\lambda$ex = 480 nm). “$\uparrow$” indicates that the fluorescence intensity shows a gradually increasing trend with the increase of concentration.

The photophysical properties of DCMOS-N and its cleavage product DCMOS were characterized. DCMOS-N exhibited a quantum yield (QY) of 27.4%, while DCMOS showed a significantly higher QY of 58.2% (2.1-fold enhancement), indicating enhanced fluorescence efficiency for sensitive detection. The extinction coefficient ($\epsilon$) of DCMOS-N was 5500 M⁻¹$\cdot$cm⁻¹, versus 37,300 M⁻¹$\cdot$cm⁻¹ for DCMOS, suggesting improved light absorption and signal-to-noise ratio. These superior photophysical properties of DCMOS, make it particularly suitable for high-contrast bioimaging (Supplementary Fig. 19).

The optimal pH range for probe DCMOS-N (10 µmol/L) in detecting biothiols (100 µmol/L) was determined based on the physiological pH conditions of living organisms. The fluorescence response of DCMOS-N was analyzed before and after interaction with biothiols across a pH range of 2 to 12. As illustrated in Fig. 5, the fluorescence intensity of DCMOS-N at 651 nm remained relatively stable under varying pH conditions. However, upon the addition of biothiols, a significant fluorescence increase was observed within the pH range of 5 to 7. The intensity showed a slight decline in weakly alkaline conditions but remained stable between pH 7 and 9. Combined with the pH-dependent test results of DCMOS (Supplementary Fig. 20). Given that most organisms maintain a physiological pH between 7.35 and 7.45, these findings indicate that probe DCMOS-N is well-suited for detecting biothiols within biological systems.

Fig. 5.

The fluorescence intensity of probe DCMOS-N (10 µmol/L) in different pH solutions after adding GSH, Cys and Hcy (10 µmol/L) ($\lambda$ex = 480 nm).

The proposed mechanism by which DCMOS-N recognizes biothiols is illustrated in Fig. 6a. Previous study has shown that the sulfhydryl group (-SH) in biothiols can undergo a nucleophilic substitution reaction with the DNBS group in the probe [21]. Specifically, the -SH group of biothiols attacks the benzene ring within the DNBS moiety, triggering a nucleophilic substitution reaction that ultimately releases SO₂ and generates the NIR-emitting dye, DCMOS. To confirm this mechanism, the reaction mixture of DCMOS-N and GSH was analyzed using LC-MS. As shown in Fig. 6b, the chromatographic peaks in the high-performance liquid spectroscopy spectra for DCMOS and DCMOS-N appeared at 14.91 and 15.95 minutes, respectively. However, after allowing DCMOS-N to react with GSH for 10 minutes, two new peaks emerged, corresponding to DCMOS (14.91 minutes) and another reaction product (12.92 minutes), while the peak for DCMOS-N disappeared. In the mass spectrometry analysis, two new peaks were observed at m/z 393.19 and 473.21 (Supplementary Fig. 21), corresponding to the NIR fluorophore DCMOS and the substitution product, respectively. These results provide strong evidence that the interaction between biothiols and the DNBS group of DCMOS-N involves a nucleophilic attack, leading to the formation of the NIR-emitting product DCMOS. This detailed mechanistic insight further validates the probe’s effectiveness in detecting biothiols.

Fig. 6.

Mechanistic study of the probe DCMOS-N for biothiols. (a) Proposed mechanism of DCMOS-N for biothiol recognition. (b) LC-MS analysis of DCMOS-N (10 µmol/L) after the addition of GSH (10 µmol/L).

Encouraged by the excellent fluorescence response of DCMOS-N toward biothiols, further studies were conducted to explore its potential for monitoring biothiols in living HeLa cells. Before proceeding with real-time cellular imaging, an MTT assay was performed to assess the cytotoxicity of DCMOS-N. As illustrated in Supplementary Figs. 22–24, HeLa, U2OS, and Jurkat cell viability remained above 80% after a 24-hour incubation with 30 µmol/L DCMOS-N, confirming its minimal cytotoxicity and suitability for live-cell imaging. Subsequently, the application of DCMOS-N for detecting biothiols in living HeLa cells was investigated using confocal microscopy. As illustrated in Fig. 7, a distinct red fluorescence signal was observed after a 30-minute incubation with the probe, indicating the presence of endogenous biothiols. However, when cells were pretreated with the well-known biothiol scavenger N-ethylmaleimide (NEM) for 30 minutes before exposure to DCMOS-N, virtually no red fluorescence was observed. To verify the capability of DCMOS-N for detecting exogenous biothiols, cells were first treated with NEM to deplete endogenous thiols. A strong red fluorescence signal was then detected after subsequent incubation with GSH and DCMOS-N for 30 minutes. Similar fluorescence responses were observed when Cys and Hcy were introduced. Moreover, no changes in cell morphology were noted throughout the experiment. These findings confirm that the probe is non-toxic and can effectively detect both endogenous and exogenous biothiols in living HeLa cells. Fluorescence imaging of cells treated with varying concentrations of biothiols and the corresponding quantitative analysis of fluorescence intensity are provided in Supplementary Figs. 25–30.

Fig. 7.

Confocal fluorescence images of endogenous and exogenous biothiols in living HeLa cells. First column: HeLa cells were incubated with probe DCMOS-N (20 µmol/L); second column: HeLa cells were incubated with NEM (200 µmol/L) and DCMOS-N (20 µmol/L); third column: NEM-pretreated HeLa cells incubated with GSH (500 µmol/L) and DCMOS-N (20 µmol/L); forth column: NEM-pretreated HeLa cells incubated with Cys (500 µmol/L) and DCMOS-N (20 µmol/L); fifth column: NEM-pretreated HeLa cells incubated with Hcy (500 µmol/L) and DCMOS-N (20 µmol/L). FL, fluorescence imaging; BF, bright field imaging. $\lambda$ex = 488 nm, $\lambda$em = 600–700 nm. Imaging scale bar = 10 µm. NEM, N-ethylmaleimide.

Additionally, experiments were conducted to demonstrate that DCMOS-N could be used to measure actual intracellular biothiol concentrations (Fig. 8). Fluorescence imaging of glutathione and cysteine at varying concentrations are shown in Supplementary Figs. 31–36, respectively. Cells were incubated with varying concentrations of biothiols for 30 minutes, followed by fluorescence imaging. The results showed a gradual increase in fluorescence intensity as biothiol concentration increased, consistent with observations in buffer solutions. These findings confirm that probe DCMOS-N is a promising tool for imaging biothiols in living cells.

Fig. 8.

Bio-imaging applications of DCMOS-N for detecting various concentrations (100 µmol/L, 200 µmol/L, 500 µmol/L) biothiols in living cells. (a) Confocal fluorescence images of different concentrations biothiols in living HeLa cells. (b) The fluorescence intensity variation with different concentrations biothiols. The data were analysed by ImageJ software (2.0, National Institutes of Health, Maryland, MD, United States). First line: GSH; second line: Cys; third line: Hcy. $\lambda$ex = 488 nm, $\lambda$em = 600–700 nm. Imaging scale bar = 10 µm. Significance was determined by the t-test (***, p 0.001).