An anthraquinone organic fluorescent probe for detecting glutathione and / or ferrous ions, and a preparation method and application thereof

Abstract

The application discloses a near-infrared fluorescent probe for detecting glutathione and / or ferrous ions, which has a structure as shown in formula I. The near-infrared fluorescent probe has the advantages of obvious spectral change, large stokes shift, high sensitivity, strong specificity, good imaging effect, low cytotoxicity and good biocompatibility, and is suitable for detecting GSH and ferroptosis in cells and biological tissues. The application also discloses application of the near-infrared fluorescent probe in preparation of reagents for detecting glutathione and / or ferrous ions. The application also discloses application of the near-infrared fluorescent probe in preparation of a diagnostic reagent kit for hepatitis, liver injury, Parkinson's disease, Alzheimer's disease and cancer or in preparation of a cell ferroptosis detection reagent kit. The application also discloses application of the near-infrared fluorescent probe in preparation of an optical imaging contrast agent.

Description

TECHNICAL FIELD

[0001] The application relates to the application of an organic small-molecule fluorescent probe in biosensing, and relates to an anthraquinone organic fluorescent probe for detecting glutathione and / or ferrous ions as well as a preparation method and application thereof. BACKGROUND

[0002] Anthraquinone is an important class of compounds, widely exists in plants and insects, and has a wide range of applications. As an important secondary metabolite of plants, anthraquinone has various biological activities, including anticancer, antibacterial and antioxidant activities. At the same time, with the deepening of the understanding of the spectral properties of anthraquinone, more and more researches are focused on the optical properties of anthraquinone, and a series of responsive fluorescent probes for detecting various analytes are developed. However, due to the restriction of the structure of anthraquinone itself, the emission wavelength of these probes is generally in the ultraviolet band. The ultraviolet wavelength has potential harm to the human body, may cause DNA damage, increase the risk of skin cancer, and at the same time, may be affected by stray light when used for detection, causing measurement error, and the signal-to-noise ratio is also relatively low, which is difficult to apply to the in vivo. Compared with the ultraviolet wavelength, the infrared wavelength has the advantages of strong penetration ability, not easy to be affected by background fluorescence and stray light, low energy and non-destructive analysis. The near-infrared band is generally divided into two regions, one region is 700nm-1100nm, and the second region is 1100-2526nm. Now the emission wavelength close to 700nm is also described as the near-infrared band to distinguish from the ultraviolet band (200-400nm). Therefore, it is very meaningful to develop a near-infrared fluorescent probe based on anthraquinone by combining the advantages of anthraquinone and the near-infrared band.

[0003] Glutathione (GSH) is the most abundant thiol in cells and tissues, and plays an important role in antioxidant defense, regulation of protein function, protein localization and stability, DNA synthesis, gene expression, cell signaling and other processes. Its content is related to various diseases such as neurodegenerative diseases [1] , cancer [2] , Parkinson's disease [3] , hepatitis and liver damage [4] , etc. In addition, ferrous ions are essential metal elements for various physiological processes in the human body, and are widely distributed in the liver and other organs, and their content is also closely related to the process of iron death. Iron death is a form of iron-dependent cell death, and studies have shown that glutathione is continuously consumed [5] during the process of cell iron death, which mainly manifests as glutathione depletion, decreased glutathione peroxidase 4 (GPX4) activity, and inability of lipid oxidants to be reduced, and then ferrous ions oxidize lipids to produce a large amount of active oxygen, resulting in cell iron death. In this process, the content of glutathione and ferrous ions is very important for cell iron death.

[0004] Therefore, it is of great significance to develop an anthraquinone-based near-infrared fluorescent probe that can be used to detect the content of glutathione and ferrous ions, and in turn enable it to specifically detect the ferroptosis process.

[0005] References:

[0006] [1] Aoyama K, Nakaki T. Impaired Glutathione Synthesis in Neurodegeneration. International Journal of Molecular Sciences, 2013: 21021-21044.

[0007] [2] Feng S, Gong S, Zheng Z, et al. Smart dual-response probe reveals an increase of GSH level and viscosity in Cisplatin-induced apoptosis and provides dual-channel imaging for tumor [J]. Sensors and Actuators B: Chemical, 2022, 351: 130940.

[0008] [3] G, Peana M, Maes M, et al. The glutathione system in Parkinson’s disease and its progression [J]. Neuroscience & Biobehavioral Reviews, 2021, 120: 470-478.

[0009] [4] Zhang W, Wang X, Li P, et al. Evaluating Hyperthyroidism-Induced Liver Injury Based on In Situ Fluorescence Imaging of Glutathione and Phosphate via Nano-MOFs Sensor [J]. Analytical Chemistry, 2020, 92(13): 8952-8958.

[0010] [5]Dong B,Li S,Wang Y,et al.Recent advance in the development of the fluorescent responsive probes for the study of ferroptosis[J].TrAC Trends inAnalytical Chemistry,2023,168:117327. Summary of the Invention

[0011] The present invention aims to provide a fluorescent probe for detecting glutathione and / or ferrous ions, and its application in ferroptosis detection. Through structural modification, this fluorescent probe enables imaging of the anthraquinone nucleus in the near-infrared band, addressing issues with ultraviolet wavelengths and showing potential for bioimaging applications.

[0012] The purpose of the present invention is achieved through the following technical solutions:

[0013] The structure of the near-infrared fluorescent probe for real-time detection of glutathione (GSH) and / or ferrous ions (denoted as AP-DNBS) is shown in Formula I:

[0014]

[0015] Another object of the present invention is to provide a method for preparing the near-infrared fluorescent probe. The synthetic route is as follows:

[0016]

[0017] The following steps are involved:

[0018] Step (a), using methanol as a reaction solvent and propiolic acid as a catalyst, compound II reacts with 4-hydroxy-3-methylbenzaldehyde to obtain compound III;

[0019] Step (b): using tetrahydrofuran as a reaction solvent and triethylamine as a catalyst, compound III reacts with 2,4-dinitrobenzenesulfonyl chloride at room temperature to obtain a near-infrared fluorescent probe for detecting glutathione as shown in formula I.

[0020] In step (a), the molar ratio of compound II to 4-hydroxy-3-methylbenzaldehyde is 1:1.0 to 1:1.3, preferably 1:1.25.

[0021] The molar ratio of the compound II to propiolic acid is 1:0.1 to 1:0.2.

[0022] The concentration of the compound II in methanol is 5-10 mg / mL.

[0023] After the reaction is completed, the mixture is cooled to room temperature, and a solid is precipitated. The solid is filtered, washed with methanol, and dried to obtain compound III.

[0024] In step (b), the molar ratio of compound III to 2,4-dinitrobenzenesulfonyl chloride is 1:1.3-1:1.6, preferably 1:1.4.

[0025] The molar ratio of compound III to triethylamine is 1:0.05-1:0.1, preferably 1:0.08.

[0026] The concentration of compound III in tetrahydrofuran is 5.9-9 mg / mL.

[0027] After the reaction is completed, the solvent is removed under reduced pressure, and the obtained residue is purified by normal phase silica gel column chromatography using dichloromethane and methanol (volume ratio = 200:1) as the eluent to obtain compound I.

[0028] The near-infrared fluorescent probe AP-DNBS uses anthraquinone (CAS: 84-65-1) as the fluorescent mother nucleus, and imidazole heterocycle is introduced at positions 1 and 2. The heterocycle is connected to a benzene ring to expand the aromatic conjugated system, so that the absorption and emission of the probe are red-shifted. At the same time, it is convenient to connect 2,4-dinitrobenzenesulfonyl through a sulfonate bond at position 23. The strong electron-withdrawing property of 2,4-dinitrobenzenesulfonyl is used to quench fluorescence. The nucleophilic substitution of GSH breaks the sulfonate bond, and the parent fluorescence is restored, so that the probe forms an “off-on” response. Figure 1 As shown in A, after the reaction of the near-infrared fluorescent probe AP-DNBS with GSH, the fluorescent quenching group 2,4-dinitrobenzenesulfonyl is replaced, and a new fluorescence is generated at 660-675 nm, which is an OFF-ON type fluorescent response. Figure 1 As shown in B, the probe retains an action pocket, which can be connected together with ferrous ions through complexation, further changing the spectroscopy results of the probe: when the excitation wavelength is 405 nm, the near-infrared fluorescent probe AP-DNBS has a strong emission peak at 545 nm, and after complexation with ferrous ions, the emission peak at 545 nm is significantly quenched.

[0029] Another object of the present application is to provide the use of the near-infrared fluorescent probe in detecting glutathione and / or ferrous ions.

[0030] Another object of the present application is to provide the use of the near-infrared fluorescent probe in preparing a reagent for detecting glutathione and / or ferrous ions.

[0031] Another object of the present application is to provide a method for detecting glutathione for non-disease diagnosis purposes, which includes qualitative detection and quantitative detection of glutathione.

[0032] Qualitative detection of glutathione, comprising the following steps:

[0033] Step S1, preparing probe stock solution: dissolving the near-infrared fluorescent probe AP-DNBS in DMSO to prepare a near-infrared fluorescent probe with a concentration of 1×10 -3 M of the probe stock solution;

[0034] Step S2, preparing the sample to be tested;

[0035] Step S3, mixing the probe stock solution and the sample to be tested, and then using PBS buffer to make up to a final concentration of 50 μM of the probe for reaction; wherein the PBS buffer has a pH of 5-8, preferably pH 7.4; the reaction temperature is 30-40°C, preferably 37°C; and the reaction time is 10-50 min, preferably 30-40 min;

[0036] Step S4, using a fluorescence spectrometer to perform fluorescence testing, and when the excitation wavelength of the reaction system is 405 nm, an emission wavelength of 675 nm is observed, indicating that the sample to be tested contains GSH.

[0037] Quantitative detection of glutathione, comprising the following steps:

[0038] Step S1, preparing probe stock solution: dissolving the near-infrared fluorescent probe AP-DNBS in DMSO to prepare a near-infrared fluorescent probe with a concentration of 1×10 -3 M of the probe stock solution;

[0039] Preparing a glutathione stock solution: dissolving glutathione in a PBS buffer with a pH of 7.4 to obtain a glutathione stock solution with a concentration of 1×10 -3 M of the glutathione stock solution;

[0040] Step S2, preparing a standard curve: mixing the probe stock solution with different volumes of GSH solution, using PBS to make up to 1 mL to obtain a series of standard curve solutions with a probe AP-DNBS concentration of 50 μM and different glutathione concentrations, incubating for reaction, and then using a fluorescence spectrometer to test the fluorescence spectrum with an excitation wavelength of 405 nm, and obtaining a standard curve with the concentration of glutathione as the abscissa and the fluorescence intensity at 675 nm as the ordinate; wherein the PBS buffer has a pH of 5-8, preferably pH 7.4; the reaction temperature is 30-40°C, preferably 37°C; and the reaction time is 10-50 min, preferably 30-40 min;

[0041] Step S3, preparing a sample to be tested; mixing the probe stock solution and the sample to be tested, and then using PBS buffer to make up to a probe concentration of 50 μM for reaction; wherein the PBS buffer has a pH of 5-8, preferably pH 7.4; the reaction temperature is 30-40°C, preferably 37°C; and the reaction time is 10-50 min, preferably 30-40 min.

[0042] Step S4, using a fluorescence spectrometer to test the reaction system obtained in step S3 for fluorescence, with an excitation wavelength of 405 nm, and obtaining the fluorescence intensity at 675 nm; and then substituting the fluorescence intensity into the standard curve obtained in step S2 to calculate the content of glutathione in the sample to be tested.

[0043] The sample to be tested can be cell supernatant.

[0044] The cell supernatant is prepared by taking cell supernatant and centrifuging at 1000 rpm for 10 min to remove particulate matter and polymers.

[0045] For qualitative detection of glutathione, the volume of the sample to be tested can be 15% of the volume of the reaction system, or the amount of the sample to be tested can be adjusted according to actual conditions.

[0046] For quantitative detection of glutathione, the volume of the sample to be tested can be 15% of the volume of the reaction system, or the amount of the sample to be tested can be adjusted according to actual conditions.

[0047] The linear range of the near-infrared fluorescent probe for detecting GSH is 0 μM-50 μM, and the detection limit is 1.02 μM.

[0048] Another object of the present application is to provide a method for detecting ferrous ions for purposes other than disease diagnosis, including qualitative detection and quantitative detection of ferrous ions.

[0049] The qualitative detection of ferrous ions comprises the following steps:

[0050] Step S1, preparing a probe stock solution: dissolving the near-infrared fluorescent probe AP-DNBS in DMSO to prepare a near-infrared fluorescent probe with a concentration of 1×10 -3 M of the probe stock solution;

[0051] Step S2, preparing a sample to be tested;

[0052] Step S3, mixing the probe stock solution and the sample to be tested, and then using PBS buffer to make up to a final probe concentration of 50 μM for reaction; wherein the PBS buffer has a pH of 5-8, preferably pH 7.4; the reaction temperature is 30-40°C, preferably 37°C;

[0053] Step S4, fluorescence test is carried out by using a fluorescence spectrometer, when the excitation wavelength of the reaction system is 405 nm, the emission wavelength change is observed at 545 nm, indicating that the sample to be tested contains ferrous ions.

[0054] The quantitative detection of glutathione GSH includes the following steps:

[0055] Step S1, preparation of probe stock solution: the near-infrared fluorescent probe AP-DNBS is dissolved in DMSO to prepare a near-infrared fluorescent probe with a concentration of 1×10 -3 M of the probe stock solution of M;

[0056] Preparation of ferrous ion mother liquor: take ferrous ions and dissolve them in PBS buffer with pH=7.4 to obtain a ferrous ion mother liquor with a concentration of 1×10 -3 M of the probe stock solution of M;

[0057] Step S2, preparation of standard curve: mix the probe stock solution with different volumes of Fe 2+ solution, dilute with PBS to 1 mL to obtain a series of standard curve solutions of the probe AP-DNBS with a concentration of 50 μM and different Fe 2+ concentrations, incubate the reaction, and then test the fluorescence spectrum by using a fluorescence spectrometer with an excitation wavelength of 405 nm, to obtain a standard curve with the concentration of Fe 2+ as the abscissa and the fluorescence intensity at 545 nm as the ordinate; wherein the pH of the PBS buffer is 5-8, preferably pH=7.4; and the reaction temperature is 30-40°C, preferably 37°C;

[0058] Step S3, preparation of sample to be tested: mix the probe stock solution with the sample to be tested, and then dilute with PBS buffer to a probe concentration of 50 μM for reaction; wherein the pH of the PBS buffer is 5-8, preferably pH=7.4; and the reaction temperature is 30-40°C, preferably 37°C;

[0059] Step S4, fluorescence test is carried out by using a fluorescence spectrometer on the reaction system obtained in step S3, with an excitation wavelength of 405 nm, to obtain the fluorescence intensity at 545 nm, which is substituted into the standard curve obtained in step S2 to calculate the content of glutathione in the sample to be tested.

[0060] The sample to be tested is the supernatant of cells with ferroptosis.

[0061] The preparation of the cell supernatant is as follows: take the cell supernatant, centrifuge at 1000 rpm for 10 min to remove particulate matter and polymers.

[0062] The cells with ferroptosis are tumor cells, such as cervical cancer cells.

[0063] In qualitative detection of ferrous ions, the volume of the sample to be tested can be 15% of the volume of the reaction system, or the amount of the sample to be tested can be adjusted according to actual conditions.

[0064] In quantitative detection of ferrous ions, the volume of the sample to be tested can be 15% of the volume of the reaction system, or the amount of the sample to be tested can be adjusted according to actual conditions.

[0065] The linear range of the near-infrared fluorescent probe for detecting ferroptosis is 200-500 muM, and the detection limit is 16.02 muM.

[0066] Another object of the present application is to provide the application of the near-infrared fluorescent probe in the preparation of a kit for detecting cell ferroptosis.

[0067] The ferroptosis is represented as Fe 2+ Abnormal. The process of ferroptosis can cause continuous depletion of glutathione and increase of ferrous ion concentration.

[0068] Another object of the present application is to provide the application of the near-infrared fluorescent probe in the preparation of an optical imaging contrast agent.

[0069] Compared with the prior art, the present application has the following beneficial effects:

[0070] 1. The near-infrared fluorescent probe has obvious spectral changes, large Stokes shift, high sensitivity, strong specificity, is not easily disturbed by biological matrix and impurities, has good imaging effect, and has low cytotoxicity and good biocompatibility, and is suitable for detection of GSH and ferroptosis process in cells and biological tissues.

[0071] 2. The preparation process of the near-infrared fluorescent probe molecule is simple, convenient to operate, and has high yield, and is suitable for large-scale production and industrial conversion. BRIEF DESCRIPTION OF DRAWINGS

[0072] Figure 1 The near-infrared fluorescent probe AP-DNBS is a "turn-on" principle diagram (A) and a complex mechanism of ferrous ions (B). Figure 1 A) and fluorescence change diagram (B). Figure 1 A) and fluorescence change diagram (B).

[0073] Figure 2 The near-infrared fluorescent probe AP-DNBS and GSH response ultraviolet (A) and fluorescence change diagram (B). Figure 2 A) and fluorescence change diagram (B). Figure 2 A) and fluorescence change diagram (B).

[0074] Figure 3 The near-infrared fluorescent probe AP-DNBS and GSH response diagram; wherein, Figure 3 A is a fluorescence intensity change diagram of the near-infrared fluorescent probe AP-DNBS after response to different concentrations of GSH. Figure 3 B is the linear relationship diagram between the near-infrared fluorescent probe AP-DNBS and GSH.

[0075] Figure 4 Fig. 4 is a diagram of the change of the fluorescence intensity of the near-infrared fluorescent probe AP-DNBS with time after GSH is added.

[0076] Figure 5 Fig. 5 is the result of the selectivity experiment of the near-infrared fluorescent probe AP-DNBS.

[0077] Figure 6 Fig. 6 is the pH stability result of the near-infrared fluorescent probe AP-DNBS.

[0078] Figure 7 Fig. 7 is the response diagram of the near-infrared fluorescent probe AP-DNBS and Fe 2+ response; wherein Figure 7 A) and fluorescence change diagram (B) Figure 7

[0079] Figure 8 Fig. 8 is the response diagram of the near-infrared fluorescent probe AP-DNBS and Fe 2+ response; wherein Figure 8 A is the fluorescence intensity change diagram of the near-infrared fluorescent probe after responding to different concentrations of Fe 2+ response; Figure 8 B is the linear relationship diagram between the near-infrared fluorescent probe AP-DNBS and Fe 2+ .

[0080] Figure 9 Fig. 9 is the toxicity experiment result of the near-infrared fluorescent probe AP-DNBS on normal cells.

[0081] Figure 10 Fig. 10 is the confocal microscope cell imaging result of the near-infrared fluorescent probe AP-DNBS. DETAILED DESCRIPTION

[0082] The technical solutions of the present application are further described below through specific embodiments.

[0083] In the present application, “M” represents mol / L, and “μM” represents μmol / L.

[0084] Example 1

[0085]

[0086] ​Compound II (0.8 mmol, 190 mg), 4-hydroxy-3-methylbenzaldehyde (1 mmol, 136 mg), propiolate (0.085 mmol, 5 μL), methanol (20 mL) were placed in a 50 mL round bottom flask with a reflux condenser, and refluxed overnight with stirring; cooled to room temperature, a solid precipitated, filtered under reduced pressure, the resulting solid was washed with methanol three times, and dried in vacuum to obtain compound III (240 mg, yield 85%) as a black solid. 1 H NMR (300 MHz, DMSO-d6) δ 12.73 (s, 1H), 10.05 (s, 1H), 8.23-8.13 (m, 3H), 8.08 (d, J = 8.4 Hz, 1H), 7.98 (d, J = 2.0 Hz, 2H), 7.89 (q, J = 5.1, 4.3 Hz, 2H), 6.92 (d, J = 8.4 Hz, 1H), 2.23 (s, 3H).13C NMR (151 MHz, DMSO-d6) δ 183.65, 182.73, 158.93, 158.71, 134.81, 134.63, 133.66, 133.54, 131.25, 127.83, 127.20, 126.62, 124.84, 121.41, 119.95, 115.25, 16.35. HR-ESI-MS m / z calcd for C 22 H 14 N2O3[M+H] + 355.1077, found 355.1085, 2.2 ppm.

[0087] Compound III (0.25 mmol, 89 mg), 2,4-dinitrobenzenesulfonyl chloride (0.35 mmol, 107.7 mg), triethylamine (0.02 mmol, 3 μL), tetrahydrofuran (10 mL) were placed in a 50 mL round bottom flask, stirred at room temperature for 3 h; the solvent was removed under reduced pressure, the resulting residue was purified by normal phase silica gel column chromatography (eluent was dichloromethane and methanol in a volume ratio = 200: 1) to obtain compound I (denoted as AP-DNBS, 61 mg, yield 42%) as a yellow powder. 1H NMR (600 MHz, DMSO-d6) δ 13.31 (s, 1H), 9.16 (d, J = 2.4 Hz, 1H), 8.67 (dd, J = 8.7, 2.3 Hz, 1H), 8.55 (s, 1H), 8.38 (d, J = 8.6 Hz, 1H), 8.30 (d, J = 8.5 Hz, 1H), 8.25 (t, J = 8.9 Hz, 2H), 8.18 - 8.11 (m, 2H), 7.95 (d, J = 5.4 Hz, 2H), 7.25 (d, J = 8.7 Hz, 1H), 2.33 (s, 3H).13C NMR (151 MHz, DMSO) δ 181.65, 181.01, 156.31, 154.38, 149.15, 147.75, 145.83, 144.68, 136.09, 135.09, 133.86, 132.91, 132.50, 130.46, 129.91, 129.04, 128.21, 127.43, 127.04, 126.81, 126.80, 125.70, 124.31, 122.85, 121.69, 119.67, 117.22, 15.83. HR-ESI-MS m / z calcd for C 28 H 16 N4O9S[M-H] - 583.0565, found 583.0523, 7.2 ppm

[0088]

[0089] The near-infrared fluorescent probe AP-DNBS takes anthraquinone as a fluorescent mother nucleus, and imidazole heterocycle is introduced at positions 13 and 14, a benzene ring is connected at position 18, and 2,4-dinitrobenzenesulfonyl is connected at position 23, which simultaneously serves as a substitution site of GSH.

[0090] Example 2

[0091] S1, preparation of a probe stock solution: 0.00294 g of the near-infrared fluorescent probe (AP-DNBS) prepared in Example 1 was accurately weighed and dissolved in 5 mL of DMSO (chromatographically pure) to fully mix and completely dissolve, to obtain a probe stock solution with a concentration of 1 x 10 - 3 M, and placed in a 4°C refrigerator for standby use.

[0092] S2, preparation of a glutathione (GSH) mother liquor: 0.00307 g of glutathione (GSH) was dissolved in 10 mL of PBS buffer (pH = 7.4) to obtain a GSH mother liquor with a concentration of 1 x 10 -3 M.

[0093] S3, in 2 mL EP tube, add the probe stock solution, GSH stock solution, and dilute to 1 mL with PBS buffer (pH = 7.4) to make the final concentration of near-infrared fluorescent probe AP-DNBS 50 μM and GSH 200 μM; replace the GSH stock solution with equal volume of PBS buffer (pH = 7.4) to make the concentration of near-infrared fluorescent probe AP-DNBS 50 μM and GSH 0 μM; mix well, incubate in 37 ℃ constant temperature water bath for 30 min, add to 1 cm quartz cuvette, test the absorption spectrum with ultraviolet visible spectrophotometer and the fluorescence spectrum with fluorescence spectrometer, and the excitation wavelength is 405 nm.

[0094] As shown in Figure 2 A, AP-DNBS has obvious ultraviolet absorption peak at 405 nm, and the absorption peak is significantly reduced after reaction with GSH. The fluorescence emission spectrum is obtained with wavelength as abscissa and fluorescence intensity as ordinate, and is shown in Figure 2 B. AP-DNBS has almost no signal at 650-675 nm, however, after reaction of AP-DNBS with GSH, the fluorescence quenching group 2,4-dinitrobenzenesulfonyl falls off, the fluorescence recovers, and there is significant fluorescence enhancement at 675 nm with 405 nm as excitation wavelength, and the stokes shift is 265 nm. The results show that the probe AP-DNBS can be used for GSH detection.

[0095] Example 3

[0096] In this example, the preparation of probe stock solution and GSH stock solution is the same as in Example 2.

[0097] In 2 mL EP tube, add the probe stock solution, GSH stock solution, and dilute to 1 mL with PBS buffer (pH = 7.4) to make the final concentration of probe AP-DNBS 50 μM and GSH 0, 5, 10, 15, 20, 25, 50, 100, 150, 200 μM respectively, mix well, incubate in 37 ℃ constant temperature water bath for 30 min, add to 1 cm quartz cuvette, and test the fluorescence spectrum with fluorescence spectrometer with 405 nm as excitation wavelength. The curve of fluorescence intensity changing with GSH concentration is obtained with GSH concentration as abscissa and fluorescence intensity at 675 nm as ordinate, and the results are shown in Figure 3 .

[0098] Figure 3 In A, the curves from bottom to top correspond to GSH concentration of 0, 5, 10, 15, 20, 25, 50, 100, 150, 200 μM respectively, and with the increase of GSH concentration, the fluorescence intensity of the system at 675 nm gradually increases. Figure 3B, in the presence of 0-50 μM GSH, there is a good linear relationship between the fluorescence intensity and the concentration of GSH, the linear equation is y = 10.587x + 1839.3, R 2 = 0.99239.

[0099] Example 4

[0100] The preparation of the probe stock solution and the GSH stock solution in this example is the same as that in Example 2.

[0101] In a 2 mL EP tube, the probe stock solution and the GSH stock solution were added, and then diluted with PBS buffer (pH = 7.4) to 1 mL, so that the final concentration of the probe AP-DNBS was 50 μM and the final concentration of GSH was 100 μM. After fully mixing, the mixture was incubated at 37°C in a constant temperature water bath for 0, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 min, respectively, and then added to a 1 cm quartz cuvette. The fluorescence spectrum of the mixture at 675 nm was tested by a fluorescence spectrometer with an excitation wavelength of 405 nm. The time was taken as the horizontal coordinate and the fluorescence intensity was taken as the vertical coordinate to obtain the kinetic experiment of the reaction between the probe and GSH.

[0102] The results are shown in Table 1. Figure 4 After the addition of GSH, the probe reacted with GSH for about 30-40 min, and the fluorescence intensity reached a relatively large value. When the reaction time was about 40 min, the fluorescence intensity reached a maximum value and remained unchanged. However, the fluorescence intensity at 40 min was not much different from that at 30 min. Therefore, 30-40 min was selected as the optimal reaction time, and 30 min was selected as the most optimal reaction time. In the subsequent experiments, the probe AP-DNBS was incubated with GSH at 37°C in a constant temperature water bath for 30 min, and then tested.

[0103] Example 5

[0104] The preparation of the probe stock solution and the GSH stock solution in this example is the same as that in Example 2.

[0105] The preparation of the interference solution: Cys (cysteine), Hcy (homocysteine), Ala (alanine), Arg (arginine), Asp (aspartic acid), Gly (glycine), Glu (glutamic acid), His (histidine), Met (methionine), Leu (leucine), Ser (serine), Tyr (tyrosine) were prepared using PBS buffer (pH = 7.4).

[0106] In 2 mL EP tube, respectively add probe stock solution, GSH mother liquor or different interference solution, add PBS buffer (pH = 7.4) to 1 mL, so that the final concentration of probe AP-DNBS is 50 μM, the concentration of GSH or other interference is 100 μM, mix well, incubate in 37 ℃ constant temperature water bath for 30 min, add to 1 cm quartz cuvette, test its fluorescence spectrum at 675 nm by fluorescence spectrometer, excitation wavelength is 405 nm; replace the same volume of GSH mother liquor with PBS buffer (pH = 7.4) as blank control (Blank).

[0107] The results are shown in Figure 5 Table 1. Except for Cys and Hcy, other interference added cannot enhance the fluorescence emission at 675 nm wavelength, and Cys and Hcy are less enhanced than GSH, and the fluorescence intensity is obviously enhanced after adding GSH, indicating that the probe AP-DNBS has good selectivity for GSH.

[0108] Example 6

[0109] The preparation of probe stock solution and GSH mother liquor in this example is the same as that in Example 2.

[0110] Add probe stock solution and GSH mother liquor in sodium phosphate-citric acid buffer with pH = 5.5, 6, 6.5, 7, 7.5 and 8, add PBS buffer (pH = 7.4) to 1 mL, so that the final concentration of probe AP-DNBS is 50 μM, the final concentration of GSH is 100 μM, mix well; take no addition of GSH as control; incubate in 37 ℃ constant temperature water bath for 30 min, add to 1 cm quartz cuvette, test its fluorescence spectrum at 675 nm by fluorescence spectrometer, excitation wavelength is 405 nm.

[0111] The results are shown in Figure 6 Table 2. In the range of pH = 5.5-8, the fluorescence intensity of the probe remains stable, indicating that the probe is stable under weak acid or weak base conditions and can respond to glutathione (GSH).

[0112] Example 7

[0113] The preparation of probe stock solution in this example is the same as that in Example 2.

[0114] Prepare Fe 2+ (ferrous ion) mother liquor: dissolve 0.00152 g of ferrous sulfate (FeSO4) in 10 mL of PBS mother liquor (pH = 7.4) to obtain Fe 2+ mother liquor, the concentration is 1 × 10 -3 M.

[0115] Add probe stock solution, Fe 2+stock solution, the final concentration of near-infrared fluorescent probe AP-DNBS was 50 μM, the final concentration of Fe 2+ stock solution, the concentration of near-infrared fluorescent probe AP-DNBS was 50 μM, the concentration of Fe 2+ stock solution, the concentration of near-infrared fluorescent probe AP-DNBS was 50 μM, the concentration of Fe 2+ was 0 μM; after fully mixed, incubated in 37℃ constant temperature water bath for 20 min, added into 1 cm quartz cuvette, the absorption spectrum was tested by ultraviolet visible spectrophotometer, and the fluorescence spectrum was tested by fluorescence spectrometer, the excitation wavelength was 405 nm.

[0116] As shown in Figure 7 A, AP-DNBS had obvious ultraviolet absorption peak at 405 nm, and after Fe 2+ reaction, the absorption peak was significantly reduced. With wavelength as abscissa and fluorescence intensity as ordinate, the fluorescence emission spectrum was obtained, as shown in Figure 7 B, AP-DNBS itself had strong emission peak at 545 nm, however, after complexation reaction with ferrous ion, the emission peak at 545 nm was significantly quenched due to the charge transfer between metal and ligand. The results showed that the probe AP-DNBS could be used for Fe 2+ detection.

[0117] Example 8

[0118] The preparation of probe stock solution was the same as that in Example 2; Fe 2+ stock solution was prepared as in Example 7.

[0119] In 2 mL EP tube, the probe stock solution, Fe 2+ stock solution, the final concentration of near-infrared fluorescent probe AP-DNBS was 50 μM, the final concentration of Fe 2+ was 50, 100, 125, 200, 250, 400, 500, 1000 μM respectively, after fully mixed, incubated in 37℃ constant temperature water bath for 20 min, added into 1 cm quartz cuvette, the fluorescence spectrum was tested by fluorescence spectrometer, the excitation wavelength was 405 nm. With Fe 2+ concentration as abscissa and fluorescence intensity at 545 nm as ordinate, the curve of fluorescence intensity change with Fe 2+ concentration was obtained, as shown in Figure 8 .

[0120] Figure 8 In A, the curves from bottom to top correspond to Fe 2+ concentration of 1000, 500, 400, 250, 200, 125, 100, 50 μM respectively, with the increase of Fe2+ With the increase of concentration, the fluorescence intensity of the system at 545nm gradually decreased. Figure 8 B, add 200 ~ 500μM Fe 2+ In the case of Fe 2+ There is a good linear relationship between the concentrations, and the linear equation is y = -109.39x + 669708.88, R 2 =0.999.

[0121] Example 9

[0122] The preparation of the probe stock solution in this example is the same as that in Example 2.

[0123] In a 2 mL EP tube, different volumes of probe stock solution were added, and then the volume was adjusted to 1 mL with serum-free culture medium to obtain probe solutions with final concentrations of 0, 500 nM, 1 μM, 5 μM, 10 μM, 20 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively.

[0124] HepG2 cells (source: ATCC, American Type Culture Collection) were cultured at a density of 6 × 10 4 The cells were seeded at a density of 1 μg / mL in a 96-well plate (with sterile PBS buffer (pH 7.4) added to the outer ring) and incubated in a 37°C, 5% CO2 incubator for 24 hours. 100 μL of the probe solution at different concentrations was added to each 96-well plate, with six replicates for each concentration. The plates were incubated in a 37°C, 5% CO2 incubator for 24 hours. The residual liquid was aspirated, and 100 μL of MTT solution (0.5 mg / mL) was added to each well. The plates were incubated in a 37°C, 5% CO2 incubator for 2 hours. The culture medium was aspirated, and 100 μL of DMSO was added to each well. The plates were shaken for 10 minutes. Once fully dissolved, the plates were analyzed using a microplate reader (detection wavelength: 490 nm).

[0125] The results are as follows Figure 9 As shown in the figure, when the probe concentration reached 200 μM, the cells still had a survival rate of more than 90%, indicating that the probe had good biocompatibility and low cytotoxicity.

[0126] Example 10

[0127] The preparation of the probe stock solution in this example is the same as that in Example 2.

[0128] Hela cells were cultured at a density of 1×10 5The cells were seeded at a density of 1 x 104 / mL into 35 mm confocal culture dishes, and cultured normally at 37°C in a 5% CO2 incubator for 24 h to allow them to adhere completely. The cells were divided into three groups: a control group (Blank), an experimental group 1 (AP-DNBS), and an experimental group 2 (Erastin + AP-DNBS). The culture medium was removed, and the control group was added with serum-free DMEM medium, the experimental group 1 was added with serum-free DMEM medium containing AP-DNBS (the probe stock solution of Example 2 was diluted with serum-free DMEM medium to a final concentration of 50 μM), and the experimental group 2 was added with serum-free DMEM medium containing AP-DNBS and the ferroptosis inducer Erastin (the probe stock solution of Example 2 was diluted with serum-free DMEM medium to a final concentration of 50 μM, and the ferroptosis inducer Erastin was diluted to a final concentration of 10 μM). The cells were incubated at 37°C in a 5% CO2 incubator for 1 h. After the incubation, the culture medium and the probe residue were removed, and the cells were washed three times with PBS buffer. Then, 200 μL of PBS was added to each dish to maintain the activity of the cells. Fluorescence imaging was performed using a confocal microscope. The probe was excited at 405 nm, and the emission wavelength range was 650-700 nm.

[0129] The results are shown in Figure 10 Compared with the control group, the cells in the experimental group 1 added with the probe AP-DNBS showed obvious fluorescence emission, and the fluorescence position overlapped well with the position of the cells photographed under bright field. The experimental group 2 added with the probe AP-DNBS and the ferroptosis inducer Erastin showed fluorescence quenching due to the depletion of glutathione (GSH) and the increase in ferrous ion concentration, which indicated that the probe could be used for specific detection of the ferroptosis process and had good potential for biological imaging applications.

Claims

1. A near-infrared fluorescent probe for detecting glutathione and / or ferrous ions having a structure as shown in Formula I:

2. A method for preparing the near-infrared fluorescent probe according to claim 1, characterized in that: The synthetic route is as follows: The following steps are involved: Step (a), using methanol as a reaction solvent and propiolic acid as a catalyst, compound II reacts with 4-hydroxy-3-methylbenzaldehyde to obtain compound III; Step (b): using tetrahydrofuran as a reaction solvent and triethylamine as a catalyst, compound III reacts with 2,4-dinitrobenzenesulfonyl chloride at room temperature to obtain a near-infrared fluorescent probe represented by formula I.

3. The method for preparing a near-infrared fluorescent probe according to claim 2, wherein: In step (a), the molar ratio of compound II to 4-hydroxy-3-methylbenzaldehyde is 1:1.0 to 1:1.3; the molar ratio of compound II to propiolic acid is 1:0.1 to 1:0.2; in step (b), the molar ratio of compound III to 2,4-dinitrobenzenesulfonyl chloride is 1:1.3 to 1:1.6; and the molar ratio of compound III to triethylamine is 1:0.05 to 1:0.

1.

4. Use of the near-infrared fluorescent probe according to claim 1 in preparing a reagent for detecting glutathione and / or ferrous ions.

5. A method for detecting glutathione, characterized in that: It includes qualitative detection and quantitative detection of glutathione; the qualitative detection of glutathione includes the following steps: Step S1, preparing a probe stock solution: dissolving the near-infrared fluorescent probe AP-DNBS according to claim 1 in DMSO to prepare a near-infrared fluorescent probe with a concentration of 1×10 -3 M probe stock solution; Step S2, preparing a sample to be tested; Step S3, mixing the probe stock solution and the sample to be tested, and then diluting the volume with PBS buffer to a final probe concentration of 50 μM, and reacting; wherein the pH of the PBS buffer is 5-8, the reaction temperature is 30-40° C., and the reaction time is 10-50 min; Step S4: performing fluorescence testing using a fluorescence spectrometer. When the excitation wavelength of the reaction system is 405 nm, an emission wavelength is observed at 675 nm, indicating that the sample to be tested contains GSH. The quantitative determination of glutathione (GSH) involves the following steps: Step S1, preparing a probe stock solution: dissolving the near-infrared fluorescent probe AP-DNBS according to claim 1 in DMSO to prepare a near-infrared fluorescent probe with a concentration of 1×10 -3 M probe stock solution; Preparation of glutathione stock solution: dissolve glutathione in PBS buffer at pH = 7.4 to obtain a concentration of 1×10 -3 M glutathione stock solution; Step S2, preparing a standard curve: mixing the probe stock solution with different volumes of GSH solution, and diluting the volume to 1 mL with PBS to obtain a series of standard curve solutions with a final concentration of 50 μM probe AP-DNBS and different glutathione concentrations, incubating the reaction, and then measuring the fluorescence spectrum using a fluorescence spectrometer with an excitation wavelength of 405 nm, with the glutathione concentration as the abscissa and the fluorescence intensity at 675 nm as the ordinate to obtain a standard curve; wherein the pH of the PBS buffer is 5-8, the reaction temperature is 30-40° C., and the reaction time is 10-50 min; Step S3, preparing the sample to be tested; mixing the probe stock solution and the sample to be tested, and then diluting the volume with PBS buffer to a final concentration of 50 μM of the probe, and reacting; wherein the pH of the PBS buffer is 5-8, the reaction temperature is 30-40° C., and the reaction time is 10-50 min; Step S4: Perform fluorescence testing on the reaction system obtained in step S3 using a fluorescence spectrometer with an excitation wavelength of 405 nm to obtain the fluorescence intensity at 675 nm. Substitute the fluorescence intensity into the standard curve obtained in step S2 to calculate the glutathione content in the sample to be tested.

6. The method for detecting glutathione according to claim 5, wherein: During the qualitative detection of glutathione, in step S3, the pH of the PBS buffer is 7.4, the reaction temperature is 37° C., and the reaction time is 30 to 40 minutes; During the quantitative detection of glutathione GSH, in step S2, the pH of the PBS buffer is 7.4, the reaction temperature is 37° C., and the reaction time is 30 to 40 minutes; in step S3, the pH of the PBS buffer is 7.4, the reaction temperature is 37° C., and the reaction time is 30 to 40 minutes.

7. A method for detecting ferrous ions, characterized in that: Including qualitative and quantitative detection of ferrous ions; The qualitative detection of ferrous ions includes the following steps: Step S1, preparing a probe stock solution: dissolving the near-infrared fluorescent probe AP-DNBS according to claim 1 in DMSO to prepare a near-infrared fluorescent probe with a concentration of 1×10 -3 M probe stock solution; Step S2, preparing a sample to be tested; Step S3, mixing the probe stock solution and the sample to be tested, and then diluting the volume with PBS buffer to a final probe concentration of 50 μM, and reacting; wherein the pH of the PBS buffer is 5-8, the reaction temperature is 30-40° C., and the reaction time is 10-20 min; Step S4: performing fluorescence testing using a fluorescence spectrometer. When the excitation wavelength of the reaction system is 405 nm, a decrease in emission intensity is observed at 545 nm, indicating that the sample to be tested contains ferrous ions. Ferrous ion Fe 2+ The quantitative detection includes the following steps: Step S1, preparing a probe stock solution: dissolving the near-infrared fluorescent probe AP-DNBS according to claim 1 in DMSO to prepare a near-infrared fluorescent probe with a concentration of 1×10 -3 M probe stock solution; Preparation of ferrous ion stock solution: dissolve ferrous ions in PBS buffer at pH 7.4 to obtain a concentration of 1×10 -3 M ferrous ion mother solution; Step S2, prepare standard curve: the probe stock solution is mixed with different volumes of Fe 2+ The solutions were mixed and diluted to 1 mL with PBS to obtain a series of standard curve solutions with a probe AP-DNBS concentration of 50 μM and different ferrous ion concentrations. The reaction was incubated and the fluorescence spectrum was measured using a fluorescence spectrometer with an excitation wavelength of 405 nm, the ferrous ion concentration as the abscissa, and the fluorescence intensity at 545 nm as the ordinate to obtain a standard curve. The pH of the PBS buffer was 5-8, the reaction temperature was 30-40° C., and the reaction time was 10-20 min. Step S3, preparing the sample to be tested; mixing the probe stock solution and the sample to be tested, and then diluting the volume with PBS buffer to a final concentration of 50 μM of the probe, and reacting; wherein the pH of the PBS buffer is 5-8, the reaction temperature is 30-40° C., and the reaction time is 10-20 min; Step S4: Perform a fluorescence test on the reaction system obtained in step S3 using a fluorescence spectrometer with an excitation wavelength of 405 nm to obtain the fluorescence intensity at 545 nm. Substitute the fluorescence intensity into the standard curve obtained in step S2 to calculate the content of ferrous ions in the sample to be tested.

8. The method for detecting ferrous ions according to claim 7, wherein: During the qualitative detection of ferrous ions, in step S3, the pH of the PBS buffer solution is 7.4, the reaction temperature is 37° C., and the reaction time is 10 to 20 minutes; Ferrous ion Fe 2+ During the quantitative detection, in step S2, the pH of the PBS buffer is 7.4, the reaction temperature is 37°C, and the reaction time is 10 to 20 minutes; in step S3, the pH of the PBS buffer is 7.4, the reaction temperature is 37°C, and the reaction time is 10 to 20 minutes.

9. Use of the near-infrared fluorescent probe according to claim 1 in the preparation of a diagnostic kit for hepatitis, liver damage, Parkinson's disease, Alzheimer's disease, or cancer, or in the preparation of a cell ferroptosis detection kit; the hepatitis, liver damage, Parkinson's disease, Alzheimer's disease, or cancer is manifested by abnormal GSH levels; the ferroptosis is manifested by Fe 2+ Abnormal levels.

10. Use of the near-infrared fluorescent probe according to claim 1 in the preparation of an optical imaging contrast agent.

Images