Anthraquinone organic fluorescent probe for detecting glutathione and / or ferrous ions as well as preparation method and application of anthraquinone organic fluorescent probe

Abstract

The invention discloses a near-infrared fluorescent probe for detecting glutathione and / or ferrous ions, and the structure of the near-infrared fluorescent probe is shown as a formula I. The near-infrared fluorescent probe disclosed by the invention is obvious in spectrum change, large in Stokes shift, high in sensitivity, strong in specificity, not easy to be interfered by biological matrixes and impurities, good in imaging effect, relatively low in cytotoxicity and relatively good in biocompatibility, and is suitable for detecting GSH and ferroptosis processes in cells and biological tissues. The invention also discloses application of the near-infrared fluorescent probe in preparation of a reagent for detecting glutathione and / or ferrous ions. The invention further discloses application of the near-infrared fluorescent probe in preparation of a kit for diagnosing hepatitis, liver injury, Parkinson's disease, Alzheimer's disease and cancer or application of the near-infrared fluorescent probe in preparation of a kit for detecting cell ferroptosis. The invention also discloses application of the near-infrared fluorescent probe in preparation of an optical imaging contrast agent. # imgabs0 #

Description

Technical Field

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

[0002] Anthraquinone is an important class of compounds that are widely found in plants and insects and have a wide range of applications. Anthraquinone, as an important secondary metabolite of plants, has a variety of biological activities, including anticancer, antibacterial and antioxidant activities. At the same time, with the deepening of the understanding of the spectral properties of anthraquinone, researchers have focused more and more research on the optical properties of anthraquinone and developed a series of responsive fluorescent probes for detecting various analytes. However, due to the structural constraints of anthraquinone itself, the emission wavelengths of these probes are generally in the ultraviolet band. Ultraviolet wavelengths are potentially harmful to the human body and may cause DNA damage and increase the risk of skin cancer. At the same time, they may be affected by stray scattered light when used for detection, resulting in measurement errors, and the signal-to-noise ratio is relatively low, making it difficult to apply in vivo. Compared with ultraviolet wavelengths, infrared wavelengths have the advantages of strong penetration, not being easily affected by background fluorescence and scattered light, and low energy, which can achieve non-destructive analysis. The near-infrared band is generally divided into two regions, the first region is 700nm ~ 1100nm, and the second region is 1100 ~ 2526nm. Now the emission wavelength close to 700nm is also generally described as the near-infrared band to distinguish it from the ultraviolet band (200 ~ 400nm). Therefore, it is very meaningful to develop near-infrared fluorescent probes 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. It plays an important role in antioxidant defense, regulation of protein function, protein localization and stability, DNA synthesis, gene expression, cell signaling, etc. Its content is related to various diseases such as neurodegenerative diseases. [1] ,cancer [2] , Parkinson's disease [3] , Hepatitis, Liver Damage [4] In addition, ferrous ions are essential metal elements for various physiological processes in the human body. They are widely distributed in the liver and other organs. At the same time, their content is also closely related to the ferroptosis process. Ferroptosis is a form of iron-dependent cell death. Studies have shown that during the process of cell ferroptosis, glutathione will be continuously consumed. [5] , which is mainly manifested by glutathione depletion, decreased activity of glutathione peroxidase 4 (GPX4), inability to reduce lipid oxides, and then ferrous ions oxidize lipids to produce a large amount of reactive oxygen species, leading to cell ferroptosis. In this process, the content of glutathione and ferrous ions is very important for cell ferroptosis.

[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 then 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 anincrease 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&BiobehavioralReviews, 2021,120:470-478.

[0009] [4] Zhang W, Wang

[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 purpose of the present invention is to provide a fluorescent probe for detecting glutathione and / or ferrous ions, and to provide its application in ferroptosis detection. The fluorescent probe realizes the imaging of the anthraquinone nucleus in the near-infrared band through structural modification, solves the problem of the ultraviolet band, and has the potential to be applied in the field of biological imaging.

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

[0013] 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 synthesis route of which 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 was completed, the mixture was cooled to room temperature, and a solid was precipitated. The solid was 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 to 1:1.6, preferably 1:1.4.

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

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

[0027] After the reaction was completed, the solvent was removed under reduced pressure, and the obtained residue was subjected to normal phase silica gel column chromatography using dichloromethane and methanol in a volume ratio of 200:1 as the eluent to purify the obtained compound I.

[0028] The near-infrared fluorescent probe AP-DNBS uses anthraquinone (CAS: 84-65-1) as the fluorescent parent nucleus, introduces imidazole heterocycles at the 1,2 positions, and connects the heterocycles to the benzene ring to expand the aromatic conjugated system, making the probe's absorption and emission red-shifted; at the same time, it is convenient to connect 2,4-dinitrobenzenesulfonyl at the 23 position through a sulfonate bond, using its strong electron-withdrawing properties to quench the fluorescence; and the nucleophilic substitution of GSH breaks the sulfonate bond, restoring the parent fluorescence, making the probe form an "off-on" response. Figure 1 As shown in A, after the near-infrared fluorescent probe AP-DNBS reacts with GSH, the fluorescence quenching group 2,4-dinitrobenzenesulfonyl is replaced, and new fluorescence is generated at 660-675nm, which is an OFF-ON type fluorescence response. Figure 1 As shown in B, the probe retains an active pocket and can be connected to ferrous ions through complexation, further changing the spectral results of the probe: when the excitation wavelength is 405nm, the near-infrared fluorescent probe AP-DNBS has a strong emission peak at 545nm. After complexation with ferrous ions, the emission peak at 545nm is significantly quenched.

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

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

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

[0032] The qualitative detection of glutathione includes the following steps:

[0033] Step S1, preparing the 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 probe stock solution;

[0034] Step S2, preparing a 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 the final concentration of the probe 50 μM, and reacting; wherein the pH of the PBS buffer is 5-8, preferably pH 7.4; the reaction temperature is 30-40° C., preferably 37° C.; the reaction time is 10-50 min, preferably 30-40 min;

[0036] Step S4: Perform 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.

[0037] The quantitative detection of glutathione includes the following steps:

[0038] Step S1, preparing the 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 probe stock solution;

[0039] 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;

[0040] Step S2, preparing a standard curve: mixing the probe stock solution with different volumes of GSH solution, and diluting the solution to 1 mL with PBS to obtain a series of standard curve solutions with a probe AP-DNBS concentration of 50 μM and different glutathione concentrations, incubating the reaction, and then testing the fluorescence spectrum with 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 to 8, preferably pH 7.4; the reaction temperature is 30 to 40° C., preferably 37° C.; the reaction time is 10 to 50 min, preferably 30 to 40 min;

[0041] 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 probe concentration of 50 μM, and reacting; wherein the pH of the PBS buffer is 5 to 8, preferably pH 7.4; the reaction temperature is 30 to 40° C., preferably 37° C.; the reaction time is 10 to 50 min, preferably 30 to 40 min;

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

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

[0044] The preparation of cell supernatant is as follows: the cell supernatant is taken and centrifuged at 1000 rpm for 10 min to remove particulate matter and polymers.

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

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

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

[0048] Another object of the present invention 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 includes the following steps:

[0050] Step S1, preparing the 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 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 diluting the solution with PBS buffer to a final concentration of 50 μM for reaction; wherein the pH of the PBS buffer is 5 to 8, preferably pH 7.4; and the reaction temperature is 30 to 40° C., preferably 37° C.;

[0053] Step S4, using a fluorescence spectrometer to perform a fluorescence test, when the excitation wavelength of the reaction system is 405 nm, a change in the emission wavelength 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, preparing the 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 probe stock solution;

[0056] 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;

[0057] Step S2, prepare standard curve: mix the probe stock solution with different volumes of Fe 2+ The solutions were mixed and the volume was adjusted to 1 mL with PBS to obtain a probe AP-DNBS concentration of 50 μM and different Fe 2+ The concentration of the series of standard curve solutions was incubated, and the fluorescence spectrum was tested with a fluorescence spectrometer, with an excitation wavelength of 405 nm and Fe 2+ The concentration is taken as the horizontal coordinate and the fluorescence intensity at 545 nm is taken as the vertical coordinate to obtain a standard curve; wherein the pH of the PBS buffer is 5 to 8, preferably pH 7.4; the reaction temperature is 30 to 40°C, preferably 37°C;

[0058] 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 probe concentration of 50 μM, and reacting; wherein the pH of the PBS buffer is 5 to 8, preferably pH 7.4; the reaction temperature is 30 to 40° C., preferably 37° C.;

[0059] Step S4: Perform fluorescence test on the reaction system obtained in step S3 using a fluorescence spectrometer, with an excitation wavelength of 405 nm, obtain the fluorescence intensity at 545 nm, substitute the fluorescence intensity into the standard curve obtained in step S2, and 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 cell supernatant is as follows: the cell supernatant is taken and centrifuged 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 the qualitative detection of ferrous ions, the volume of the sample to be tested can be 15% of the volume of the reaction system, and the amount of the sample to be tested can also be adjusted according to actual conditions.

[0064] When quantitatively detecting ferrous ions, the volume of the sample to be tested can be 15% of the volume of the reaction system, and the amount of the sample to be tested can also be adjusted according to actual conditions.

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

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

[0067] The ferroptosis is expressed as Fe 2+ Abnormal levels. The ferroptosis process leads to a continuous depletion of glutathione and an increase in ferrous ion concentrations.

[0068] Another object of the present invention is to provide the use of the near-infrared fluorescent probe in the preparation of optical imaging contrast agents.

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

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

[0071] 2. The near-infrared fluorescent probe molecule preparation process of the present invention is simple, easy to operate, high in yield, and suitable for large-scale production and industrial transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 The schematic diagram of the “off-on” principle of the near-infrared fluorescent probe AP-DNBS ( Figure 1 A) and the complexation mechanism of ferrous ions ( Figure 1 B).

[0073] Figure 2 The near-infrared fluorescent probe AP-DNBS responds to UV ( Figure 2 A) and fluorescence change diagram ( Figure 2 B).

[0074] Figure 3 is the response diagram of near-infrared fluorescent probe AP-DNBS and GSH; Figure 3 A is the fluorescence intensity change spectrum of the near-infrared fluorescent probe AP-DNBS in 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 This is a graph showing the change in fluorescence intensity over time of the near-infrared fluorescent probe AP-DNBS after adding GSH.

[0076] Figure 5 These are the experimental results of the selectivity of the near-infrared fluorescent probe AP-DNBS.

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

[0078] Figure 7 AP-DNBS is a near-infrared fluorescent probe that binds to Fe 2+ Response to UV ( Figure 7 A) and fluorescence change diagram ( Figure 7 B)

[0079] Figure 8 AP-DNBS is a near-infrared fluorescent probe that binds to Fe 2+ Response plot; where Figure 8 A is the near-infrared fluorescent probe and different concentrations of Fe 2+ Fluorescence intensity change spectrum after response; Figure 8 B is the near-infrared fluorescent probe AP-DNBS and Fe 2+ Linear relationship diagram between .

[0080] Fig. 9 These are the experimental results of the toxicity of the near-infrared fluorescent probe AP-DNBS to normal cells.

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

[0082] The technical solution of the present invention is further described below through specific implementation methods.

[0083] In the present invention, "M" means mol / L, and "μM" means μmol / L.

[0084] Example 1

[0085]

[0086] Compound II (0.8mmol, 190mg), 4-hydroxy-3-methylbenzaldehyde (1mmol, 136mg), propiolic acid (0.085mmol, 5μL) and methanol (20mL) were placed in a 50mL round-bottom flask equipped with a reflux condenser and refluxed overnight with stirring; cooled to room temperature, solid precipitated, and filtered under reduced pressure. The obtained solid was rinsed three times with methanol and dried in vacuo to obtain compound III (240mg, yield 85%) as a black solid. 1 H NMR(300MHz,DMSO-d6)δ12.73(s,1H),10.05(s,1H),8.23-8.13(m,3H),8.08(d,J=8.4Hz,1H ),7.98(d,J=2.0Hz,2H),7.89(q,J=5.1,4.3Hz,2H),6.92(d,J=8.4Hz,1H),2.23(s,3H).13C NMR (151MHz, DMSO-d6) δ183.65,182.73,158.93,158.71,134.81,134.63,133.66,133. 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, 2.2ppm.

[0087] Compound III (0.25mmol, 89mg), 2,4-dinitrobenzenesulfonyl chloride (0.35mmol, 107.7mg), triethylamine (0.02mmol, 3μL) and tetrahydrofuran (10mL) were placed in a 50mL round-bottom flask and stirred at room temperature for 3h; the solvent was removed under reduced pressure, and the residue was purified by normal phase silica gel column chromatography (eluent: dichloromethane and methanol in a volume ratio of 200:1) to obtain compound I (denoted as AP-DNBS, 61mg, yield 42%) as a yellow powder. 1H NMR (600MHz, DMSO-d6) δ13.31(s,1H),9.16(d,J=2.4Hz,1H),8.67(dd,J=8.7,2.3Hz,1H),8.55(s,1H),8.38(d,J=8.6Hz,1H), 8.30(d,J=8.5Hz,1H),8.25(t,J=8.9Hz,2H),8.18-8.11(m,2H),7.95(d,J=5.4Hz,2H),7.25(d,J=8.7Hz,1H),2.33(s,3H).13C NMR (151MHz, DMSO) δ181.65,181.01,156.31,154.38,149.15,147.75,145.83,144.68,136.09,135.09,133.86,132.91,132.50,13 0.46,129.91,129.04,128.21,127.43,127.04,126.81,126.80,125.70,124.31,122.85,121.69,119.67,117.22,15.83.HR-ESI-MS m / z calcd for C 28 H 16 N4O9S[MH] - 583.0565, found583.0523, 7.2ppm

[0088]

[0089] The near-infrared fluorescent probe AP-DNBS uses anthraquinone as the fluorescent mother core, introduces imidazole heterocycles at positions 13 and 14, connects to a benzene ring at position 18, and connects to a 2,4-dinitrobenzenesulfonyl group at position 23, which also serves as a substitution site for GSH.

[0090] Example 2

[0091] S1. Preparation of probe stock solution: accurately weigh 0.00294 g of the near-infrared fluorescent probe (AP-DNBS) prepared in Example 1, dissolve it in 5 mL of DMSO (chromatographic grade), and mix thoroughly to completely dissolve it to obtain a probe stock solution with a concentration of 1×10 - 3 M, placed in a 4℃ refrigerator for later use.

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

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

[0094] like Figure 2 As shown in A, AP-DNBS has an obvious ultraviolet absorption peak at 405nm, and the absorption peak is significantly reduced after reacting with GSH. With wavelength as the horizontal axis and fluorescence intensity as the vertical axis, the fluorescence emission spectrum is obtained, see Figure 2 B, AP-DNBS has almost no signal at 650-675nm. However, after AP-DNBS reacts with GSH, the fluorescence quenching group 2,4-dinitrobenzenesulfonyl falls off, and the fluorescence is restored. With 405nm as the excitation wavelength, there is a significant fluorescence enhancement at 675nm, and the Stokes shift is measured to be 265nm. The results show that the probe AP-DNBS can be used for GSH detection.

[0095] Example 3

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

[0097] Add the probe stock solution and GSH mother solution to a 2mL EP tube, and dilute to 1mL with PBS buffer (pH=7.4), so that the final concentration of the probe AP-DNBS is 50μM, and the final concentration of GSH is 0, 5, 10, 15, 20, 25, 50, 100, 150, 200μM, respectively. Mix thoroughly, incubate in a 37℃ constant temperature water bath for 30min, add to a 1cm quartz cuvette, and test the fluorescence spectrum with a fluorescence spectrometer, with an excitation wavelength of 405nm. With GSH concentration as the horizontal axis and the fluorescence intensity at 675nm as the vertical axis, a curve of fluorescence intensity changing with GSH concentration is obtained, as shown in the following figure. Figure 3 shown.

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

[0099] Example 4

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

[0101] Add the probe stock solution and GSH mother solution to a 2mL EP tube, and dilute to 1mL with PBS buffer (pH=7.4), so that the final concentration of the probe AP-DNBS is 50μM and the final concentration of GSH is 100μM, mix well, incubate in a 37℃ constant temperature water bath for 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50min, add to a 1cm quartz cuvette, and test its fluorescence spectrum at 675nm with a fluorescence spectrometer, with an excitation wavelength of 405nm. With time as the abscissa and fluorescence intensity as the ordinate, the kinetic experiment of the reaction between the probe and GSH is obtained.

[0102] The results are as follows Figure 4 As shown in the figure, after adding GSH, the probe and GSH reacted for about 30 to 40 minutes, and the fluorescence intensity reached a maximum value. When the reaction was about 40 minutes, the fluorescence intensity reached the maximum value and remained unchanged. However, the fluorescence intensity of the reaction for 40 minutes was not much different from that of the reaction for 30 minutes. Therefore, 30 to 40 minutes was selected as the better reaction time, and 30 minutes was the optimal reaction time. In the subsequent experiments, the probe AP-DNBS was selected to be incubated with GSH in a constant temperature water bath at 37°C for 30 minutes before testing.

[0103] Example 5

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

[0105] Preparation of interfering substance solution: Take Cys (cysteine), Hcy (homocysteine), Ala (alanine), Arg (arginine), Asp (aspartic acid), Gly (glycine), Glu (glutamic acid), His (histidine), Met (methionine), Leu (leucine), Ser (serine), and Tyr (tyrosine) and prepare them in PBS buffer (pH = 7.4).

[0106] In a 2 mL EP tube, the probe stock solution, GSH mother solution or different interfering substance solutions were added respectively, and the volume was fixed to 1 mL with PBS buffer (pH = 7.4), so that the final concentration of the probe AP-DNBS was 50 μM, and the concentration of GSH or other interfering substances was 100 μM. The tubes were fully mixed, incubated in a 37°C constant temperature water bath for 30 min, added to a 1 cm quartz cuvette, and the fluorescence spectrum at 675 nm was tested with a fluorescence spectrometer, with an excitation wavelength of 405 nm; an equal volume of GSH mother solution was replaced with PBS buffer (pH = 7.4) as a blank control (Blank).

[0107] The results are as follows Figure 5 As shown, except for Cys and Hcy, other interfering substances added could not enhance the fluorescence emitted at a wavelength of 675 nm, and Cys and Hcy were less enhanced than GSH. The fluorescence intensity was significantly enhanced after the addition of GSH, indicating that the probe AP-DNBS has good selectivity for GSH.

[0108] Example 6

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

[0110] The probe stock solution and GSH mother solution were added to disodium hydrogen phosphate-citrate buffer at pH = 5.5, 6, 6.5, 7, 7.5, and 8, respectively, and the volume was adjusted 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, and the mixture was thoroughly mixed; no addition of GSH was used as a control; the mixture was incubated in a constant temperature water bath at 37°C for 30 min, added to a 1 cm quartz cuvette, and its fluorescence spectrum at 675 nm was tested using a fluorescence spectrometer, with an excitation wavelength of 405 nm.

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

[0112] Example 7

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

[0114] Formulated Fe 2+ (Ferrous ion) mother solution: Dissolve 0.00152 g of ferrous sulfate (FeSO4) in 10 mL of PBS mother solution (pH = 7.4) to obtain Fe 2+ Stock solution, concentration is 1×10 -3 M.

[0115] Add the probe stock solution, Fe 2+The stock solution was diluted to 1 mL with PBS buffer (pH = 7.4) to make the final concentration of the near-infrared fluorescent probe AP-DNBS 50 μM and Fe 2+ The final concentration was 200 μM; Fe was replaced with an equal volume of PBS buffer (pH = 7.4). 2+ The concentration of near-infrared fluorescent probe AP-DNBS was 50 μM, Fe 2+ The concentration was 0 μM; the mixture was fully mixed, incubated in a constant temperature water bath at 37°C for 20 min, added to a 1 cm quartz cuvette, and the absorption spectrum was tested with a UV-visible spectrophotometer and the fluorescence spectrum was tested with a fluorescence spectrometer, with an excitation wavelength of 405 nm.

[0116] like Figure 7 As shown in A, AP-DNBS has an obvious UV absorption peak at 405nm and Fe 2+ After the reaction, the absorption peak decreased significantly. With wavelength as the horizontal axis and fluorescence intensity as the vertical axis, the fluorescence emission spectrum was obtained, see Figure 7 B, AP-DNBS itself has a strong emission peak at 545nm. However, after the complexation reaction with ferrous ions, the emission peak at 545nm is significantly quenched due to the charge transfer effect between the metal and the ligand. The results show that the probe AP-DNBS can be used for Fe 2+ Detection.

[0117] Example 8

[0118] The preparation of the probe stock solution in this embodiment is the same as that in embodiment 2; Fe 2+ The preparation of the mother liquor is the same as in Example 7.

[0119] Add the probe stock solution, Fe 2+ The mother solution was diluted to 1 mL with PBS buffer (pH = 7.4) to make the final concentration of the probe AP-DNBS 500 μM. 2+ The final concentrations were 50, 100, 125, 200, 250, 400, 500, and 1000 μM, respectively. They were fully mixed, incubated in a 37°C constant temperature water bath for 20 min, added to a 1 cm quartz cuvette, and the fluorescence spectrum was tested using a fluorescence spectrometer with an excitation wavelength of 405 nm. 2+ The fluorescence intensity at 545 nm is taken as the ordinate and the fluorescence intensity is taken as the horizontal axis. 2+ The concentration change curve is as follows Figure 8 shown.

[0120] Figure 8 In A, the curves from bottom to top correspond to Fe 2+ The concentrations of Fe2+ With the increase of concentration, the fluorescence intensity of the system at 545nm gradually decreased. Figure 8 B, add 200-500 μM 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 6×10 4 / mL density was inoculated into a 96-well plate (sterile PBS buffer with pH = 7.4 was added to the outer circle) and cultured in a 37°C, 5% CO2 incubator for 24 hours. 100μL of the above-mentioned probe solution of different concentrations was respectively aspirated and added to the 96-well plate, and 6 replicates were made for each concentration, and incubated in a 37°C, 5% CO2 incubator for 24 hours. The residual liquid was aspirated, 100μL MTT solution (0.5mg / mL) was added to each well, and incubated in a 37°C, 5% CO2 incubator for 2 hours, the culture medium was aspirated, 100μL DMSO was added to each well, and shaken for 10 minutes. After it was fully dissolved, it was detected using an enzyme reader (the detection wavelength was 490nm).

[0125] The results are as follows Fig. 9 As shown, when the probe concentration reached 200 μM, the cell survival rate was still above 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 1×10 5The cells were inoculated into a 35 mm confocal culture dish at a density of 100 μg / mL and cultured in a 37°C, 5% CO2 incubator for 24 hours to completely adhere to the wall. The cells were divided into three groups: control group (Blank), experimental group 1 (AP-DNBS), and experimental group 2 (Erastin+AP-DNBS); the culture medium was aspirated, serum-free DMEM culture medium was added to the control group, serum-free DMEM culture medium containing AP-DNBS was added to the experimental group 1 (the probe stock solution in Example 2 was diluted with serum-free DMEM culture medium to a final concentration of 50 μM), and serum-free DMEM culture medium containing AP-DNBS and ferroptosis inducer Erastin was added to the experimental group 2 (the probe stock solution in Example 2 was diluted with serum-free DMEM culture medium to a final concentration of 50 μM, and the final concentration of ferroptosis inducer Erastin was 10 μM), and incubated in a 37°C, 5% CO2 incubator for 1 hour; after the incubation, the culture medium and probe residues were aspirated, and the cells were rinsed three times with PBS buffer, and 200 μL PBS was added to each dish to maintain cell activity. Fluorescence imaging was detected using a confocal microscope. The probe was excited at 405 nm and the emission wavelength range was collected from 650 to 700 nm.

[0129] The results are as follows Fig.10 As shown in the figure, 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 cell position photographed in the bright field state. In the experimental group 2, which added the probe AP-DNBS and the ferroptosis inducer Erastin at the same time, the depletion of glutathione (GSH) and the increase in the concentration of ferrous ions led to fluorescence quenching. This phenomenon shows that the probe can be used to specifically detect the ferroptosis process and has good potential for bioimaging 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 shown in formula I.

3. The method for preparing the near-infrared fluorescent probe according to claim 2, characterized in that: In step (a), the molar ratio of the compound II to 4-hydroxy-3-methylbenzaldehyde is 1:1.0 to 1:1.3; the molar ratio of the compound II to propiolic acid is 1:0.1 to 1:0.2; in step (b), the molar ratio of the compound III to 2,4-dinitrobenzenesulfonyl chloride is 1:1.3 to 1:1.6; the molar ratio of the 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 described in 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 solution with PBS buffer to a final concentration of 50 μM for reaction; wherein the pH of the PBS buffer is 5 to 8, the reaction temperature is 30 to 40° C., and the reaction time is 10 to 50 min; Step S4, using a fluorescence spectrometer to perform a fluorescence test, 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 detection of glutathione GSH includes the following steps: Step S1, preparing a probe stock solution: dissolving the near-infrared fluorescent probe AP-DNBS described in 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 solution to 1 mL with PBS to obtain a series of standard curve solutions with a final concentration of 50 μM of the probe AP-DNBS and different glutathione concentrations, incubating the reaction, and then testing the fluorescence spectrum with a fluorescence spectrometer, with an excitation wavelength of 405 nm, the concentration of glutathione as the horizontal axis, and the fluorescence intensity at 675 nm as the vertical axis, to obtain a standard curve; wherein the pH of the PBS buffer solution is 5 to 8, the reaction temperature is 30 to 40° C., and the reaction time is 10 to 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 test on the reaction system obtained in step S3 using a fluorescence spectrometer, with an excitation wavelength of 405 nm, obtain the fluorescence intensity at 675 nm, substitute the fluorescence intensity into the standard curve obtained in step S2, and calculate the content of glutathione in the sample to be tested.

6. The method for detecting glutathione according to claim 5, characterized in that: 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 min; 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 min; 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 min.

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 described in 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 using PBS buffer to make the final concentration of the probe 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, using a fluorescence spectrometer to perform a fluorescence test, 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 described in 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: mix the probe stock solution with different volumes of Fe 2+ The solutions were mixed and fixed to 1 mL with PBS to obtain a series of standard curve solutions with a concentration of 50 μM of the probe AP-DNBS and different ferrous ion concentrations, incubated for reaction, and then tested for fluorescence spectrum with a fluorescence spectrometer, with an excitation wavelength of 405 nm, the concentration of ferrous ions as the abscissa, and the fluorescence intensity at 545 nm as the ordinate, to obtain a standard curve; wherein the pH of the PBS buffer solution is 5 to 8, the reaction temperature is 30 to 40° C., and the reaction time is 10 to 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, using a fluorescence spectrometer to perform a fluorescence test on the reaction system obtained in step S3, with an excitation wavelength of 405 nm, obtaining the fluorescence intensity at 545 nm, substituting the fluorescence intensity into the standard curve obtained in step S2, and calculating the content of ferrous ions in the sample to be tested.

8. The method for detecting ferrous ions according to claim 7, characterized in that: During the qualitative detection of ferrous ions, 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 min; 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 min; 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 min.

9. The use of the near-infrared fluorescent probe of claim 1 in the preparation of a diagnostic kit for hepatitis, liver injury, Parkinson's disease, Alzheimer's disease, or cancer, or in the preparation of a cell ferroptosis detection kit; the hepatitis, liver injury, 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