A near-infrared fluorescent probe for detecting gsh and a preparation method and biological imaging application thereof

Abstract

The application discloses a kind of near-infrared fluorescent probe for detecting GSH and its preparation method and biological imaging application, belong to biological analysis technical field, the fluorescent probe NRh-BZ is by being decorated to oxygen heterocyclic skeleton with hemicyanine, increase conjugated structure, realize the detection of GSH in near-infrared light region, belong to off-on type fluorescent probe, with high selectivity, sensitivity and low cytotoxicity, its emission wavelength is 746nm, detection limit is as low as 46.8nM, can be applied to the detection imaging of GSH in living cell MCF-7 and rat liver tissue, and tissue penetration depth reaches 150 μm.The fluorescent probe NRh-BZ prepared in the application can quantitatively detect GSH in various biological systems.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical technology, and in particular relates to a near-infrared fluorescent probe for detecting GSH, its preparation method, and its application in bioimaging. Background Technology

[0002] Glutathione (GSH) is a tripeptide compound composed of cysteine, glutamic acid, and glycine. It is the most abundant non-protein biothiol in cells, with concentrations in the millimolecular range. It exists primarily in the reduced thiol form (GSH) and the oxidized dithiolated form (GSSG) within cells, and their levels maintain a dynamic balance. GSH plays a crucial role in regulating intracellular redox homeostasis. Furthermore, it activates various enzymes, thereby promoting carbohydrate, fat, and protein metabolism and influencing cellular metabolic processes. It is an important intracellular regulator of metabolism, participating in intracellular antioxidant processes, maintaining intracellular signaling, and gene regulation. Abnormal levels of GSH are associated with many diseases, including slow growth, liver damage, cancer, AIDS, and neurodegenerative diseases. Therefore, developing a highly sensitive and selective method for detecting glutathione is of great significance.

[0003] In recent years, fluorescence analysis based on fluorescent probes has been widely used in various biological research fields due to its advantages such as simple operation, high sensitivity, real-time spatial imaging, and non-destructive effects on samples. However, most of the reported GSH probes are concentrated in the visible light region (Liu, J.; Sun, Y.; Zhang, H.; et al. Chem. Sci., 2014, 5(12): 3183-3188. Liu, G.; Chen W., Xu Z., et al. Org. Biomol. Chem.; 2018, 16(30): 5517-5523.). Excitation by visible light sources can easily lead to photobleaching and phototoxicity, and is easily affected by background fluorescence in biological tissues, limiting its application in biology and clinical diagnosis. Therefore, developing fluorescent probes with emission wavelengths in the near-infrared region is crucial for applications in the biological field. However, we note that although there are some reports on near-infrared fluorescent probes for GSH detection, there is still a problem that they cannot distinguish between GSH, Cys, and Hcy, thus failing to achieve GSH-specific detection (Li, R.; Kassaye, H.; Pan, Y.; et al. Biomater. Sci., 2020, 8(21): 5994-6003. Hu, Q.; Yu, C.; Xia, X.; et al. Biosens. Bioelectron., 2016, 81(07): 341-348. Xu, S.; Zhou, J., Dong, X., et al. Analytica. Chimica. Acta., 2019, 1074(29): 123-130.). In addition, the response speed of existing probes needs to be improved. For example, the response speeds of the relevant probes reported in existing technologies Xie, JY, Li, CY, Li, YF, Fei, J., et al. Anal. Chem. 2016, 88(19): 9746-9752. and Tong, L.; Qian, YJ Mater. Chem. B. 2018, 6(12): 1791-1798. are 100s and 50s respectively, and there is still a need to improve the sensitivity.

[0004] Therefore, developing a highly selective and sensitive near-infrared fluorescent probe for detecting GSH in vitro and in vivo, with good biocompatibility, is of great significance in the fields of biology and disease diagnosis. Summary of the Invention

[0005] To address the aforementioned issues, this invention, based on the excellent photophysical and photochemical properties of xanthracene, provides a near-infrared fluorescent probe for detecting GSH, its preparation method, and its bioimaging applications. This invention employs a strategy of expanding the π-conjugated system of xanthracene, achieving detection wavelengths in the near-infrared region by modifying hemicyanine with the xanthracene skeleton. By introducing 4-bromomethylbenzaldehyde, which recognizes SH, a "closed-closed" naked-eye near-infrared fluorescent probe NRh-BZ for GSH detection was successfully designed and synthesized. This probe exhibits high sensitivity for GSH detection, distinguishing between GSH, Cys, and Hcy, and is unaffected by interference from other amino acids, demonstrating excellent selectivity. More importantly, this probe can also be applied to the detection of GSH in living cells and tissues.

[0006] One of the solutions provided by this invention:

[0007] One fluorescent probe, NRh-BZ, is an oxane fluorescent probe based on hemicyanine modification.

[0008] This invention connects the xanthene skeleton with a hemicyanine fluorophore as a novel fluorescent probe, which achieves detection through fluorescence generation and quenching, exhibiting high detection sensitivity. The fluorescent probe NRh-BZ proposed in this invention uses the xanthene fluorophore as the parent nucleus, exhibiting low cytotoxicity and good biocompatibility, and can be widely used in living cells and tissues.

[0009] The structural formula of the fluorescent probe NRh-BZ is:

[0010] The fluorescent probe NRh-BZ proposed in this invention recognizes GSH based on substitution reactions and intramolecular amino-induced spironolactam ring opening. Because the spironolactam ring is closed, the fluorescent probe NRh-BZ does not exhibit fluorescence. In a GSH environment, the thiol group at one end of the GSH ring undergoes a substitution reaction with the -Br group on the probe, forming an unstable intermediate. The GSH provides an acidic environment, and the intermediate undergoes hydrolysis to yield the hydrazide hydrolysis product. The spironolactam ring then opens, enhancing fluorescence, thereby enabling rapid detection of GSH content in complex biological samples.

[0011] The second technical solution provided by this invention:

[0012] A method for preparing a near-infrared fluorescent probe NRh-BZ for detecting GSH:

[0013] Under ice bath conditions, 4-diethylaminoketo acid was added in portions to a concentrated sulfuric acid solution of cyclohexanone, heated to react, cooled, and then perchloric acid solution was added. The mixture was filtered, washed, and dried to obtain intermediate M1. The method for expanding the oxanthracene π-conjugated system was as follows: intermediate M1 and Fisher's aldehyde were added to acetic anhydride, stirred at room temperature, and after the reaction was terminated, the crude product was obtained by vacuum distillation. After purification, intermediate M2 was obtained. PyBOP (benzotriazol-1-yl-oxytripyrrolidinephosphide hexafluorophosphate) and hydrazine hydrate were added sequentially to a dichloromethane solution of intermediate M2, stirred again at room temperature, and the crude product was obtained by vacuum distillation. After purification, intermediate M3 was obtained. Intermediate M3 and 4-bromomethylbenzaldehyde were refluxed in ethanol, and the crude product was obtained by vacuum distillation. After purification, the fluorescent probe NRh-BZ was obtained.

[0014] This invention prepares intermediate M1 from cyclohexanone and 4-diethylaminoketo acid; by expanding the π-conjugated system of oxanthracene, a GSH recognition group is introduced by aldehyde-amine condensation reaction with 4-bromomethylbenzaldehyde, and a near-infrared fluorescent probe NRh-BZ for detecting GSH is prepared.

[0015] Compounds with an oxanthracene skeleton structure generally possess excellent photophysical properties, including high molar extinction coefficient, high fluorescence quantum yield, and good photostability. This invention synthesizes a novel fluorescent probe based on a hemicyanine-modified oxanthracene skeleton by introducing a 4-bromomethylbenzaldehyde recognition group. Detection is achieved through the generation and quenching of fluorescence, exhibiting high detection sensitivity.

[0016] More preferably, the molar ratio of cyclohexanone to 4-diethylaminoketo acid is 1:1; the heating reaction temperature is 90°C and the time is 2.5 h; the washing is performed three times with cold water; and the concentration of perchloric acid is 70 wt%.

[0017] More preferably, the molar ratio of intermediate M1 to Fisher's aldehyde is 1:1; and the stirring reaction at room temperature is carried out for 0.5 h.

[0018] More preferably, the molar ratio of intermediate M2, PyBOP and hydrazine hydrate is 2.15:2.68:15.8; and the second stirring reaction at room temperature is carried out for 4 hours.

[0019] More preferably, the molar ratio of intermediate M3 to 4-bromomethylbenzaldehyde is 0.734:1.1; the reflux reaction time is 2-3 hours, during which the reaction process is monitored by TLC.

[0020] More preferably, the purification of intermediates M2 and M3 and the fluorescent probe NRh-BZ is performed by column chromatography, with the eluent being a mixture of dichloromethane and ethanol at different concentration gradients; the volume ratio of dichloromethane to ethanol in the eluent for intermediate M2 ranges from 20:1 to 200:1; the volume ratio of dichloromethane to ethanol in the eluent for intermediate M3 and the near-infrared fluorescent probe NRh-BZ for detecting GSH ranges from 50:1 to 200:1. Since the impurities and products have similar polarities, purification is more difficult. During column chromatography purification, attention should be paid to the eluent ratio, and slow elution with low polarity is recommended.

[0021] The "on-off" ring properties of the fluorescent probe NRh-BZ proposed in this invention are affected by acidity and alkalinity. During synthesis, purification, preservation and spectroscopic experiments, the pH should be maintained within the range of 6 to 8.5, and the influence of acidic or alkaline environments should be avoided.

[0022] The third technical solution provided by this invention:

[0023] The above-mentioned fluorescent probe NRh-BZ is used in bioimaging, specifically for detecting glutathione in solution; for preparing a fluorescent imaging agent to detect glutathione in living cells; and for preparing a fluorescent imaging agent to detect glutathione in tissues.

[0024] The beneficial effects of this invention are:

[0025] The fluorescent probe NRh-BZ proposed in this invention undergoes substitution and hydrolysis reactions with GSH, inducing the opening of the lactam ring to generate a metabolite. This is an off-on fluorescence reaction. Within the range of 0–4 μM, the fluorescence intensity shows a good linear relationship with the concentration of GSH (R>0.99), and its detection limit is as low as 46.8 nM. This indicates that the fluorescent probe NRh-BZ can be used as a chemical sensor for the quantitative detection of GSH and has good practicality.

[0026] The fluorescent probe NRh-BZ proposed in this invention has low cytotoxicity, good biocompatibility, and can be widely used in living cells and tissues, with a tissue penetration depth of up to 150 μm.

[0027] The fluorescent probe NRh-BZ proposed in this invention is not easily affected by the biological matrix during the detection process of specific probe reactions. The response intensity of the probe remains almost unchanged before and after the addition of other amino acids (Cys, Hcy, Thr, Phe, Met, Ala, Ile, Pro, Gly, His, Ser, Val, Arg), indicating that the fluorescent probe NRh-BZ has good anti-interference ability for the detection of GSH and can be used for the detection of GSH in complex biological samples in various biological systems. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0029] Figure 1 The synthesis flowchart for the fluorescent probe NRh-BZ prepared in this invention is shown below;

[0030] Figure 2 The 1H-NMR spectrum of the fluorescent probe NRh-BZ prepared in Example 1;

[0031] Figure 3 The 13C-NMR spectrum of the fluorescent probe NRh-BZ prepared in Example 1;

[0032] Figure 4 The ESI-HRMS spectrum of the fluorescent probe NRh-BZ prepared in Example 1;

[0033] Figure 5 The UV-Vis absorption spectra of the fluorescent probe NRh-BZ (10 μM) prepared in Example 1 before and after responding to GSH (20 μM) in an ethanol / water (1:1 v / v, pH=7.4) system;

[0034] Figure 6 The fluorescence emission spectra of the fluorescent probe NRh-BZ (10 μM) prepared in Example 1 before and after responding to GSH (20 μM) in an ethanol / water (1:1 v / v, pH=7.4) system;

[0035] Figure 7 The UV absorption spectra of the fluorescent probe NRh-BZ (10 μM) prepared in Example 1 when 0–20 μM GSH was gradually added to an ethanol / water (1:1 v / v, pH = 7.4) system;

[0036] Figure 8 The fluorescence emission spectrum of the fluorescent probe NRh-BZ (10 μM) prepared in Example 1 when 0–20 μM GSH was gradually added to an ethanol / water (1:1 v / v, pH = 7.4) system.

[0037] Figure 9 The linear relationship between the emission intensity of the fluorescent probe NRh-BZ (10 μM) prepared in Example 1 at 746 nm and the GSH concentration (0–4 μM) is shown in the graph.

[0038] Figure 10 A schematic diagram illustrating the mechanism of action of the fluorescent probes NRh-BZ and GSH prepared in this invention;

[0039] Figure 11The UV absorption spectra of the fluorescent probe NRh-BZ (10 μM) prepared in Example 1 were obtained by adding biothiols and various amino acids (GSH, Cys, Hcy, Thr, Phe, Met, Ala, Ile, Pro, Gly, His, Ser, Val, Arg) to an ethanol / water (1:1 v / v, pH=7.4) system.

[0040] Figure 12 The fluorescence emission spectra of the fluorescent probe NRh-BZ (10 μM) prepared in Example 1 were obtained by adding biothiols and various amino acids (GSH, Cys, Hcy, Thr, Phe, Met, Ala, Ile, Pro, Gly, His, Ser, Val, Arg) to an ethanol / water (1:1 v / v, pH=7.4) system.

[0041] Figure 13 The fluorescence probe NRh-BZ (10 μM) prepared in Example 1 was tested for its resistance to GSH (20 μM) in the presence of biothiols and amino acids (20 μM) (Cys, Hcy, Thr, Phe, Met, Ala, Ile, Pro, Gly, His, Ser, Val, Arg).

[0042] Figure 14 The fluorescence intensity of the fluorescent probe NRh-BZ (10 μM) prepared in Example 1 changes over time in the presence of GSH (20 μM). The system is ethanol / water (1:1 v / v, pH=7.4).

[0043] Figure 15 The graph shows the effect of pH on the fluorescent probe NRh-BZ prepared in Example 1.

[0044] Figure 16 The results of the cytotoxicity assay are shown on the x-axis, which represents the concentration of the fluorescent probe, and the y-axis represents the cell viability.

[0045] Figure 17 The image shows a confocal image of the fluorescent probe NRh-BZ prepared in Example 1, where (a1)-(c1) represent endogenous imaging, (a2)-(c2) represent the clearing of intracellular GSH, and (a3)-(c3) represent exogenous imaging.

[0046] Figure 18The image shows a fluorescence image of rat liver tissue containing the fluorescent probe NRh-BZ prepared in Example 1. (a) The tissue section was incubated with the probe NRh-BZ (10 μM) for 1 h; (b) The tissue section was pretreated with NEM (2 mM) for 1 h and then incubated with NRh-BZ (10 μM) for 1 h; (c) Confocal z-scan imaging was performed at different depths of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, and 150 μm, with λex = 633 nm. Detailed Implementation

[0047] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0048] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0049] The room temperature in this invention refers to 25±2℃.

[0050] Example 1: Synthesis process of fluorescent probe NRh-BZ

[0051] Synthesis of intermediate M1: Cyclohexanone (1.98 mL, 9.6 mmol) was added dropwise to concentrated sulfuric acid (20.0 mL) under ice bath conditions. Then, 4-diethylaminoketo acid (3.00 g, 9.6 mmol) was added in portions with vigorous stirring. After the addition was complete, the mixture was reacted at 90 °C for 2.5 h, then cooled and poured into crushed ice (150.0 g). Perchloric acid (2.0 mL, 70 wt%) was then added, and a red precipitate immediately appeared. The precipitate was filtered and washed three times with cold water and then dried in air to obtain a red solid, which was intermediate M1, with a yield of approximately 94%. No purification was required for the next step.

[0052] Synthesis of intermediate M2: Intermediate M1 (2.5 g, 6.65 mmol) and Fisher's aldehyde (1.34 g, 6.65 mmol) were directly dissolved in acetic anhydride (35.0 mL) and stirred at room temperature for 0.5 h. The resulting mixture was then immediately placed at 0 °C to terminate the reaction. The crude product was obtained by vacuum distillation and purified by column chromatography using a concentration gradient of ethanol-dichloromethane (volume ratio range of 200:1 to 20:1 v / v) to obtain a black-green solid, which was intermediate M1, with a yield of 50 ± 5%.

[0053] Synthesis of intermediate M3: Intermediate M2 (1.2 g, 2.15 mmol) was dissolved in dichloromethane (25 mL), and then PyBOP (1.4 g, 2.68 mmol) and hydrazine hydrate (2.4 mL, 15.8 mmol) were added sequentially. The mixture was stirred at room temperature for 4 h. The reaction solution was then distilled under reduced pressure to obtain the crude product. The crude product was purified by column chromatography using a concentration gradient of ethanol-dichloromethane (volume ratio range of 200:1 to 50:1 v / v) as eluent to obtain a yellow powder, which was intermediate M3, with a yield of 45 ± 5%.

[0054] Intermediate M3 (0.42 g, 0.734 mmol) and 4-bromomethylbenzaldehyde (0.21 g, 1.1 mmol) were refluxed in ethanol for 3 h. The reaction progress was monitored by TLC until the spot of intermediate M3 disappeared. The resulting reaction solution was then distilled under reduced pressure to obtain the crude product. The crude product was purified by column chromatography using a concentration gradient of ethanol-dichloromethane (volume ratio range of 200:1 to 50:1 v / v) as eluent to obtain a yellow powder, which is the near-infrared fluorescent probe NRh-BZ for the detection of GSH, with a yield of 10%.

[0055] The synthetic route diagram of the above preparation method is shown below. Figure 1 .

[0056] The HNMR spectrum information of intermediate M2 is as follows: 1 H NMR (400MHz, CDCl3) δ (ppm) 8.57 (d, J = 14.0Hz, 1H), 8.25 (d, J = 7.8Hz, 1H), 7.71 (t, J = 7.4Hz, 1H),7.60(t,J=7.7Hz,1H),7.39(dd,J=12.6,7.5Hz,3H),7.22(d,J=7.7Hz,1H),7.15(t,J=6 .7Hz,2H),6.70(d,J=9.0Hz,1H),6.60(d,J=10.2Hz,2H),6.06(d,J=13.4Hz,1H),3.67(s,3H ), 3.51 (d, J = 7.1Hz, 4H), 2.64 (d, J = 16.1Hz, 2H), 2.27 (d, J = 15.0Hz, 2H), 1.37 ~ 1.12 (m, 14H).

[0057] The CNMR spectrum information of intermediate M2 is as follows: 13C NMR (101MHz, CDCl3) δ (ppm) 173.12, 168.22, 163.29, 155.94, 152.30, 152. 03,142.92,141.99,140.62,136.14,133.36,131.83,129.38,129.15,128. 73,128.27,125.07,122.47,122.24,121.13,115.98,113.56,112.31,110. 49,99.33,95.88,49.13,45.22,31.58,28.50,26.62,24.22,20.47,12.51.

[0058] The HNMR spectrum information of intermediate M3 is as follows: 1 HNMR (400MHz, CDCl3) δ (ppm) 7.90 (d, J = 7.1Hz, 1H), 7.61 ~ 7.39 (m, 5H), 7.18 (dd, J = 1 5.1,7.4Hz,3H),6.84(t,J=7.1Hz,1H),6.60(t,J=8.6Hz,1H),6.35(d,J=8.8Hz,2H), 6.29(d,J=8.9Hz,1H),5.37(d,J=12.4Hz,1H),3.35(dd,J=14.2,7.1Hz,4H),3.15(s ,3H),2.55(d,J=34.9Hz,4H),1.74~1.69(m,6H),1.25(s,2H),1.18(t,J=7.0Hz,6H).

[0059] The CNMR spectrum information of intermediate M3 is as follows: 13 C NMR (101MHz, CDCl3) δ (ppm) 166.3, 157.8, 153.2, 149.63, 148.58, 145.37, 138.88, 132.3, 130.81, 128.21, 127.7, 125.7, 123.48, 122.9, 121.5 ,119.6,119.30,116.53,111.2,108.48,105.69,102.73,102.2,98.0,9 2.02, 67.84, 45.4, 44.4, 29.1, 28.55~28.11, 25.36, 23.0, 22.3, 12.52.

[0060] ¹H NMR spectrum information of the fluorescent probe NRh-BZ: 1H NMR (400MHz, CDCl3) δ (ppm) 8.67 (d, J = 6.6Hz, 1H), 7.95 (d, J = 7.4Hz, 2H), 7.61 (t, J = 11.3Hz, 3H), 7.53 (d, J = 9.0Hz, 2 H),7.47~7.43(m,1H),7.24~7.15(m,3H),6.86(t,J=7.5Hz,1H),6.66~6.59(m,1H),6.47(t,J=6.6Hz,1H),6.38(s,1H ),6.24(d,J=8.4Hz,1H),5.38(d,J=12.7Hz,1H),4.47(d,J=8.5Hz,2H),3.48(dd,J=13.9,6.9Hz,3H),3.33(dd,J=14 .0,7.0Hz,4H),3.15(s,2H),2.49(d,J=7.6Hz,2H),1.77(d,J=9.7Hz,6H),1.40~1.36(m,2H),1.17(d,J=7.4Hz,6H), such as Figure 2 As shown.

[0061] CNMR spectrum information of fluorescent probe NRh-BZ: 13 C NMR (101MHz, CDCl3) δ165.45,157.79,156.55,154.45,154.07,152.31,151.74,150.45,149.23~148.66 ,146.85,145.51,138.99,134.76,133.32,129.90,129.71,129.57,129.20~128.66,128.66~127.12,126 .40,124.28,123.48,121.68,120.63,119.82,119.46,108.68~108.61,108.36,105.83,104.66,97.93,92.20,72.42,65.87,45.03,32.05,30.27~29.50,27.35,22.82,15.32,14.24,12.70.ESI-HRMS:Theoretical m / z[C 48 H 46 N4O2Br+H] + :753.2726, measured: 753.2791, as Figure 3 As shown.

[0062] The ESI-HRMS image of the fluorescent probe NRh-BZ is shown below. Figure 4 Among them, the theoretical m / z[C 48 H46 N4O2Br+H] + : 753.2726, measured: 753.2791.

[0063] Example 2

[0064] Fluorescence effect experiment of the fluorescent probe NRh-BZ prepared in Example 1:

[0065] 1. UV absorption and fluorescence emission experiments of the fluorescent probe NRh-BZ:

[0066] The fluorescent probe NRh-BZ (10 μM) was added to an ethanol / water (1:1 v / v, pH=7.4) system, and the absorption and fluorescence spectra were measured using a UV-3600i UV-Vis spectrophotometer and an FLS1000 steady-state / transient fluorescence spectrometer.

[0067] GSH solutions of 0, 0.25, 0.5, 1, 1.5, 2, and 20 μM were added to the fluorescent probe NRh-BZ (10 μM), and its absorption and emission spectra were measured.

[0068] Figure 5 The UV-Vis absorption spectrum of the fluorescent probe NRh-BZ (10 μM) prepared in Example 1 in a mixture of ethanol / water (1:1 v / v, pH=7.4) and GSH (20 μM).

[0069] Figure 6 Fluorescence emission spectra of the fluorescent probe NRh-BZ (10 μM) prepared in Example 1 in an ethanol / water (1:1 v / v, pH=7.4) system and in a mixture with GSH (20 μM).

[0070] Figure 7 The UV absorption spectra of the fluorescent probe NRh-BZ (10 μM) prepared in Example 1 when 0–20 μM GSH was gradually added to an ethanol / water (1:1 v / v, pH = 7.4) system.

[0071] Figure 8 The fluorescence emission spectrum of the fluorescent probe NRh-BZ (10 μM) prepared in Example 1 when 0–20 μM GSH was gradually added to an ethanol / water (1:1 v / v, pH = 7.4) system.

[0072] Figure 9 The linear relationship between the emission intensity of the fluorescent probe NRh-BZ (10 μM) prepared in Example 1 at 746 nm and the GSH concentration (0-4 μM).

[0073] Depend on Figure 5-9It can be seen that the emission wavelength of the fluorescent probe NRh-BZ is in the near-infrared region; the Stokes shift reaches 27 nm; the fluorescence intensity at the maximum emission peak has a good linear relationship with the GSH concentration (0-4 μM), and its detection limit is calculated to be as low as 46.8 nM.

[0074] A schematic diagram illustrating the mechanism of action of the fluorescent probes NRh-BZ and GSH prepared in this invention is shown below. Figure 10 .

[0075] 2. Selectivity experiment of fluorescent probe NRh-BZ for GSH fluorescence detection:

[0076] The fluorescent probe NRh-BZ prepared in Example 1 was prepared into a 10 μM test solution in an ethanol / water (1:1 v / v, pH=7.4) system. Then, 20 μM of biothiol and amino acids (GSH, Cys, Hcy, Thr, Phe, Met, Ala, Ile, Pro, Gly, His, Ser, Val, Arg) were added respectively. After shaking and standing, mixed solutions of fluorescent probe NRh-BZ and the above different amino acids were obtained. The UV absorption spectrum and fluorescence emission spectrum of each mixed solution were tested.

[0077] Figure 11 The UV absorption spectra of the fluorescent probe NRh-BZ (10 μM) prepared in Example 1 were obtained by adding biothiols and various amino acids (GSH, Cys, Hcy, Thr, Phe, Met, Ala, Ile, Pro, Gly, His, Ser, Val, Arg) to an ethanol / water (1:1 v / v, pH=7.4) system.

[0078] Figure 12 The fluorescence emission spectra of the fluorescent probe NRh-BZ (10 μM) prepared in Example 1 were obtained by adding biothiols and various amino acids (GSH, Cys, Hcy, Thr, Phe, Met, Ala, Ile, Pro, Gly, His, Ser, Val, Arg) to an ethanol / water (1:1 v / v, pH=7.4) system.

[0079] Depend on Figure 11 and 12 It can be seen that the pure probe has no obvious absorption peak at 719 nm and the emission peak is also weak. However, after adding GSH, the absorption intensity at 719 nm and the fluorescence intensity at 746 nm are significantly enhanced. When other amino acids are added to the test solution, the fluorescence intensity is almost unchanged compared with the probe, indicating that the fluorescent probe NRh-BZ prepared in this invention has high selectivity for the detection of GSH.

[0080] 3. Anti-interference experiment of fluorescent probe NRh-BZ for GSH fluorescence detection:

[0081] Take 13 5mL EP tubes, mix the 10μM fluorescent probe NRh-BZ with GSH, and perform fluorescence spectroscopy. After the test, add other substances (Cys, Hcy, Thr, Phe, Met, Ala, Ile, Pro, Gly, His, Ser, Val, Arg) at the same concentration as GSH, mix well, and then perform fluorescence spectroscopy again under the same test conditions.

[0082] Figure 13 The image shows the anti-interference experiment of the fluorescent probe NRh-BZ (10 μM) prepared in Example 1 against GSH (20 μM) in the presence of various amino acids (20 μM) (Cys, Hcy, Thr, Phe, Met, Ala, Ile, Pro, Gly, His, Ser, Val, Arg). Figure 13 It can be seen that the fluorescent probe NRh-BZ showed almost no change in response intensity before and after the addition of the above amino acids, indicating that the fluorescent probe NRh-BZ prepared in this invention has good anti-interference ability for the detection of GSH and can be used for the detection of GSH in complex biological samples.

[0083] 4. Response time of fluorescent probe NRh-BZ to GSH fluorescence detection:

[0084] The fluorescent probe NRh-BZ prepared in Example 1 was prepared into a fluorescent probe stock solution with a molar concentration of 10 μM in an ethanol / water (1:1 v / v, pH=7.4) system. 20 μM GSH was added to the solution, and after gentle stirring, the GSH was allowed to diffuse on its own. The change in fluorescence intensity at 746 nm over time was then detected.

[0085] Figure 14 The fluorescence intensity of the fluorescent probe NRh-BZ (10 μM) prepared in Example 1 changes over time in the presence of GSH (20 μM). The system is ethanol / water (1:1 v / v, pH = 7.4).

[0086] Depend on Figure 14 It can be seen that fluorescence appears within 5 seconds after adding GSH (20 μM) to the fluorescent probe NRh-BZ (10 μM), indicating that the probe reacts with GSH at this time. The fluorescence intensity increases significantly with time, reaching its maximum at around 20 seconds, after which the fluorescence intensity no longer changes. This indicates that the fluorescent probe NRh-BZ prepared in this invention has a faster GSH recognition response than most GSH fluorescent sensors and has higher sensitivity for GSH recognition.

[0087] 5. Effect of pH on the detection of GSH by the fluorescent probe NRh-BZ:

[0088] Distilled water was adjusted to pH 5–10 using 0.5–1.0 M NaOH / HCl solution, and then mixed with an equal volume of ethanol to obtain ethanol / water (1:1, v / v) mixed solution systems with different pH values. An appropriate amount of fluorescent probe NRh-BZ stock solution was then added to these systems to prepare 10 μM fluorescent probe NRh-BZ solutions with different pH values. A scatter plot of probe fluorescence intensity versus pH was obtained. Figure 15 The effect of pH on the fluorescent probe NRh-BZ prepared in Example 1 is shown in the figure. It can be seen that the absorption intensity of the fluorescent probe NRh-BZ before and after interaction with GSH remains basically unchanged within the pH range of 6.0 to 8.5, indicating that the fluorescent probe NRh-BZ prepared in this invention can recognize GSH under physiological pH conditions and can be applied to the detection of GSH in living cells and tissues.

[0089] Example 3

[0090] Imaging experiments of exogenous and endogenous GSH cells using the fluorescent probe NRh-BZ prepared in Example 1.

[0091] 1. Cytotoxicity assay of fluorescent probe NRh-BZ on MCF-7 cells:

[0092] The cytotoxicity of the probe was evaluated using the MTT assay. MCF-7 cells were seeded in 96-well cell culture plates (1×10⁶ cells / wells). -4 Cells were incubated in 10 wt% fetal bovine serum (FBS) and DMEM medium in a 5% CO2 incubator for 24 h. After the cells showed good growth, a negative control group (cells with fluorescent probe NRh-BZ) was set up. At the same time, the cells were incubated with five fluorescent probe NRh-BZ solutions of different concentrations (0, 1.25, 2.5, 5, 10, 20 μM) for 24 h. Then, the cells were incubated with 5 mg / mL MTT for another 4 h. After incubation, the original medium was discarded, 150 μL DMSO was added, and the OD value at 570 nm was detected by a microplate reader.

[0093] Cell viability results as follows Figure 16 The cytotoxicity test results show that the cell survival rate is above 96%, proving that the fluorescent probe NRh-BZ prepared in this invention is non-toxic to MCF-7 cells and can be widely used in live cells and tissues.

[0094] 2. Staining and imaging experiments of live cells using the fluorescent probe NRh-BZ:

[0095] MCF-7 cells were cultured in a 5% CO2 incubator with 10 wt% fetal bovine serum (FBS) and DMEM medium. Triple aliquots were seeded into 35 mm laser confocal microscopy culture dishes and cultured for 24 h. One aliquot was first washed three times with PBS, then incubated for 30 min in a 5% CO2 incubator with DMEM medium containing the fluorescent probe NRh-BZ (10 μM), followed by three more washes with PBS. Immediately afterward, endogenous imaging was performed under a laser confocal microscope. The second aliquot was first incubated with 500 μM DMEM medium... After incubation in DMEM medium containing N-ethylmaleimide (NEM) (a bio-thiol blocker) for 30 min, the cells were washed three times with PBS, then incubated with the fluorescent probe NRh-BZ (10 μM) for 30 min, followed by three washes with PBS. Immediately after incubation, the cells were imaged under a laser confocal microscope using the same testing conditions as the previous group. The final sample consisted of MCF-7 cells pretreated with 500 μM N-ethylmaleimide (NEM) and co-incubated with the fluorescent probe NRh-BZ (10 μM) for 30 min, washed three times with PBS, then incubated with 100 μM MSH for 30 min, washed three times with PBS, and immediately imaged under a laser confocal microscope using the same testing conditions as the previous two groups. Cell imaging experiments were performed using a STELLARIS5 super-resolution laser confocal microscope with an excitation wavelength of 633 nm. Results are as follows: Figure 17 Cellular confocal imaging of the fluorescent probe NRh-BZ prepared in Example 1, where (a1)-(c1) represent endogenous GSH imaging, (a2)-(c2) represent GSH clearance within cells, and (a3)-(c3) represent exogenous GSH imaging. In vitro staining and imaging experiments on live cells show that the fluorescent probe NRh-BZ prepared in this invention can detect not only endogenous GSH but also exogenous GSH.

[0096] Example 4

[0097] Fluorescence imaging experiment of rat liver tissue sections using the fluorescent probe NRh-BZ prepared in Example 1:

[0098] Tissue sections were prepared from rat liver tissue, and one side of the rat liver tissue was cut flat using a vibrating blade microtome. One tissue section was placed in the culture medium of the fluorescent probe NRh-BZ (10 μM) and incubated at 37°C for 1 h. After washing three times with PBS, images were created. Another tissue section was pretreated with 2 mM NEM for 1 h, washed three times with PBS, and then added to the culture medium of the fluorescent probe NRh-BZ (10 μM). It was incubated at 37°C for 1 h and washed three times with PBS before imaging. Fluorescence intensity changes with scanning depth were acquired using the z-scan mode of a Zeiss LSM880 laser confocal scanning microscope, with an excitation wavelength of 633 nm.

[0099] The results are as follows Figure 18 The fluorescence imaging of rat liver tissue prepared by the fluorescent probe NRh-BZ in Example 1 is shown, wherein (a) the tissue section was co-incubated with the fluorescent probe NRh-BZ (10 μM) for 1 h; (b) the tissue section was pretreated with NEM (2 mM) for 1 h, and then incubated with NRh-BZ (10 μM) for 1 h; (c) confocal z-scan imaging at different depths of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, and 150 μm, λex = 633 nm. This indicates that in living tissue, the fluorescent probe NRh-BZ prepared in this invention has good tissue penetration and GSH sensing capabilities, with a penetration depth of up to 150 μm.

[0100] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A near-infrared fluorescent probe NRh-BZ for detecting GSH, characterized in that, The chemical structural formula of the near-infrared fluorescent probe NRh-BZ is: .

2. A method for preparing the fluorescent probe NRh-BZ according to claim 1, characterized by, The specific steps are: adding 4-diethylaminoketonic acid into concentrated sulfuric acid solution of cyclohexanone under ice bath condition, heating reaction, adding perchloric acid solution after cooling, filtering, washing and drying the precipitate to obtain intermediate M1; adding intermediate M1 and Fischer's aldehyde into acetic anhydride, stirring reaction at room temperature, distilling the crude product under reduced pressure after reaction termination, and obtaining intermediate M2 after purification; adding PyBOP and hydrazine hydrate into dichloromethane solution of intermediate M2 in sequence, stirring reaction at room temperature again, distilling the crude product under reduced pressure, and obtaining intermediate M3 after purification; refluxing reaction of intermediate M3 and 4-bromomethylbenzaldehyde in ethanol, distilling the crude product under reduced pressure, and obtaining fluorescent probe NRh-BZ after purification; The chemical structural formula of the intermediate M1 is: ; The chemical structural formula of the intermediate M2 is: ; The chemical structural formula of the intermediate M3 is: .

3. The preparation method according to claim 2, characterized in that, The molar ratio of cyclohexanone and 4-diethylaminoketonic acid is 1:1; the temperature of heating reaction is 90℃, and the time is 2.5h; the washing is cold water washing for 3 times.

4. The preparation method according to claim 2, characterized in that, The molar ratio of intermediate M1 and Fischer's aldehyde is 1:1; the time of stirring reaction at room temperature is 0.5h.

5. The preparation method according to claim 2, characterized in that, The molar ratio of intermediate M2, PyBOP and hydrazine hydrate is 2.15:2.68:15.8; the time of stirring reaction at room temperature again is 4h.

6. The method of claim 2, wherein, The molar ratio of intermediate M3 and 4-bromomethylbenzaldehyde is 0.734:1.1; the time of refluxing reaction is 2-3h.

7. The preparation method according to claim 2, characterized in that, The purification of intermediate M2, intermediate M3 and fluorescent probe NRh-BZ all adopts column chromatography.

8. Application of fluorescent probe NRh-BZ of claim 1 in preparing fluorescent imaging agent for detecting glutathione in living cells or in preparing fluorescent imaging agent for detecting glutathione in tissues.

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