A near-infrared ferrous ion fluorescent probe and its preparation and application

Abstract

The present invention discloses a near-infrared iron ion fluorescent probe and its preparation and application, belonging to the fields of analytical chemistry and biotechnology. The excitation and emission wavelengths of the fluorescent probe disclosed in the present invention are both located in the near-infrared region, and it has excellent selectivity for ferrous ions, and can achieve rapid detection of endogenous and exogenous ferrous ions in cells, can effectively avoid the interference of sample autofluorescence, and improve the signal-to-noise ratio; the probe can be located in mitochondria, which is crucial for studying the dynamics and functions of ferrous ions in mitochondria. The probe can image endogenous and exogenous ferrous ions in tumor cells in real time and quickly, showing good application prospects of biofluorescence imaging, providing a powerful tool for tumor research, helping to reveal the role of ferrous ions in tumors, evaluate the efficacy of targeted therapy, and optimize treatment plans. It has potential clinical value in tumor diagnosis, treatment monitoring, and prognosis evaluation, and at the same time shows important technical effects and broad application prospects in biomedical research and clinical diagnosis.

Description

Technical Field

[0001] The present invention belongs to the field of analytical chemistry and biotechnology, and in particular relates to a near-infrared ferrous ion fluorescent probe and its preparation and application. Background Art

[0002] As an essential trace element for the human body, iron plays an indispensable role in the body. Its redox activity is key to many biological processes (such as oxygen transport, electron transfer and enzyme catalysis). 2+ An imbalance in iron homeostasis, particularly in organelles like mitochondria and lysosomes, is closely linked to the onset and progression of a variety of diseases, including neurodegenerative diseases and cancer. When iron homeostasis is disrupted within cells, it leads to the release of harmful reactive oxygen species (ROS), which in turn triggers oxidative stress and cell damage. This imbalance is closely linked to the onset and progression of a variety of diseases, including neurodegenerative diseases and cancer.

[0003] To accurately monitor intracellular ferrous ion concentrations, scientists have developed a variety of detection methods. Traditional methods, such as electrochemical detection, atomic absorption spectroscopy (AAS), and inductively coupled plasma mass spectrometry (ICPMS), while highly accurate, are complex to operate and require expensive equipment, limiting their application in real-time monitoring and bioimaging.

[0004] In recent years, with the rapid development of bioluminescence technology, fluorescent probes have gradually become a powerful tool for detecting intracellular ferrous ions due to their advantages such as ease of operation, low cost, and intuitive detection results. Fluorescent probes specifically bind to ferrous ions, producing a significant change in fluorescence signal, thereby achieving highly sensitive and selective detection of ferrous ions. Although existing fluorescent probes have made some progress in detecting ferrous ions, some problems still exist, such as insufficient selectivity and the need for improved sensitivity. Therefore, the development of a highly selective and sensitive fluorescent probe for ferrous ions that can be used for real-time imaging in vivo is of great significance for gaining a deeper understanding of the biological functions of iron, revealing the relationship between iron imbalance and disease, and guiding the diagnosis and treatment of related diseases. Summary of the Invention

[0005] In response to the problems of insufficient selectivity and improved sensitivity of existing ferrous ion fluorescent probes during the detection process, the present invention aims to provide a ferrous ion fluorescent probe with high selectivity, high sensitivity and real-time imaging in organisms, so as to achieve accurate monitoring of ferrous ions and further understand the biological functions of iron and its relationship with diseases.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a near-infrared ferrous ion fluorescent probe, the structure of which is as follows:

[0008] .

[0009] The present invention provides a method for preparing the above-mentioned near-infrared ferrous ion fluorescent probe, comprising:

[0010] S1, under argon protection, 5-bromosalicylaldehyde was mixed with cesium carbonate, dissolved and reacted with 2-bromocyclohex-1-ene-1-carbaldehyde, extracted, the organic layer was dried, and purified by column chromatography to prepare compound 1;

[0011] The structural formula of the compound 1 is: ;

[0012] ;

[0013] S2, under argon protection, compound 1 in S1 was mixed with 1-ethyl-2-methylquinolinium iodide, a solvent was added for reaction, the mixture was filtered under reduced pressure, and the mixture was dried to obtain compound 2;

[0014] The structural formula of the compound 2 is: ;

[0015] ;

[0016] S3, under nitrogen protection, the compound 2 obtained in S2, 4-amino-2,2,6,6-tetramethylpiperidinyl 1-oxyl radical, cesium carbonate, 2-dicyclohexylphosphino-2'-(N,N-dimethylamino)biphenyl and trisdibenzylideneacetone dipalladium were mixed for reaction, extracted, dried, and purified by column chromatography to obtain a near-infrared ferrous ion fluorescent probe;

[0017] .

[0018] In S1, the molar ratio of 5-bromosalicylaldehyde, cesium carbonate, and 2-bromocyclohex-1-ene-1-carbaldehyde is 4-5:10-12:1, the reaction temperature is 24°C-26°C, and the reaction time is 10-14 h; the solvent used for the extraction is water and dichloromethane, and the mobile phase used for the column chromatography purification is petroleum ether and ethyl acetate in a volume ratio of 20:1.

[0019] In S2, the molar ratio of the compound 1 to 1-ethyl-2-methylquinolinium iodide is 1:1-2, the solvent is acetic anhydride, the reaction temperature is 100°C-120°C, and the reaction time is 8 h-12 h.

[0020] In S3, the molar ratio of the compound 2, 4-amino-2,2,6,6-tetramethylpiperidinyl 1-oxyl free radical, cesium carbonate, 2-dicyclohexylphosphino-2'-(N,N-dimethylamino)biphenyl and trisdibenzylideneacetone dipalladium is 1:5~6:2~3:0.1~0.2:0.1~0.2, the reaction temperature is 90℃~110℃, and the reaction time is 5 h~7 h.

[0021] In S3, the extraction uses water and dichloromethane as solvents, and the column chromatography purification uses dichloromethane and methanol in a volume ratio of 45:1 as mobile phases.

[0022] The present invention provides the use of the near-infrared ferrous ion fluorescent probe in preparing a reagent for measuring, screening or detecting ferrous ions, wherein the ferrous ions are endogenous or exogenous ferrous ions in cells.

[0023] The present invention provides application of the near-infrared ferrous ion fluorescent probe in cell fluorescence imaging.

[0024] The present invention provides a method for detecting the presence of ferrous ions in a sample or determining the ferrous ion content in a sample, comprising:

[0025] (1) using the above kit to contact the near-infrared ferrous ion fluorescent probe with the sample to form a fluorescent compound;

[0026] (2) Determine the fluorescence properties of the fluorescent compound.

[0027] Compared with the prior art, the present invention achieves the following technical effects:

[0028] The near-infrared ferrous ion fluorescent probe provided by the present invention has excitation and emission wavelengths both in the near-infrared region. This characteristic makes the probe more penetrating in biological tissues, reduces light scattering and background noise, and improves the clarity and depth of imaging. It has a large Stokes shift, which means that the wavelength difference between the excitation light and the emission light is large, which helps to reduce interference from the excitation light and improve the signal-to-noise ratio of the signal. It can be localized in mitochondria, which are the main energy producers in cells and important sites for ferrous ion metabolism, and is of great significance for studying the dynamics and function of ferrous ions in mitochondria. It can be used together with other commercial dyes without generating signal interference, which increases its application flexibility in complex biological environments. It can achieve real-time and rapid cell fluorescence imaging of endogenous ferrous ions in tumor cells, indicating that it has good application prospects in the field of biological fluorescence imaging.

[0029] The preparation method of the near-infrared ferrous ion fluorescent probe provided by the present invention is prepared through a multi-step organic synthesis reaction, the reaction conditions are mild and easy to control, and the purification steps are simple, which is suitable for large-scale production; the raw materials are readily available, the cost is low, and it is suitable for large-scale production, which is conducive to the practical application and promotion of the probe.

[0030] The application of the near-infrared ferrous ion fluorescent probe provided by the present invention can realize real-time and rapid cell fluorescence imaging of endogenous ferrous ions in tumor cells, providing a powerful tool for the study of tumor growth, metastasis and treatment. Through imaging, the molecular mechanisms of tumor growth, metastasis and treatment can be deeply understood, which helps to reveal the role of ferrous ions in tumor development. It can be used to evaluate the efficacy of tumor targeted therapy and provide a basis for the optimization and adjustment of tumor treatment plans. It has potential clinical application value in the early diagnosis, staging, treatment monitoring and prognosis evaluation of tumors. It also has important technical effects and broad application prospects in biomedical research and clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the high-resolution mass spectrometry (HRMS) spectrum of the probe DHX-Fe of the present invention;

[0032] Figure 2 is the electron paramagnetic resonance (EPR) spectrum of the probe DHX-Fe of the present invention;

[0033] Figure 3 The electron paramagnetic resonance (EPR) spectra of the probe DHX-Fe of the present invention before and after the response to ferrous ions;

[0034] Figure 4 The UV absorption spectra of the probe DHX-Fe of the present invention before and after responding to ferrous ions (0-10eq);

[0035] Figure 5 Fluorescence emission spectra of the probe DHX-Fe of the present invention before and after responding to (0-10eq) ferrous ions;

[0036] Figure 6 Fluorescence intensity changes of the probe DHX-Fe of the present invention at different times after the addition of ferrous ions (Ex / Em: 610nm / 690nm, slitband: 3nm / 3nm);

[0037] Figure 7 This is the fluorescence spectrum of the probe DHX-Fe of the present invention for the selectivity and interference of ferrous ions;

[0038] Figure 8 This is a laser confocal imaging image (A2780 cells) of the fluorescent probe of the present invention recognizing exogenous ferrous ions, with a scale bar of 20 μM;

[0039] Figure 9 This is a laser confocal imaging image (SKOV3 cells) of the fluorescent probe of the present invention recognizing endogenous ferrous ions induced by Erastin; scale bar 20 μM;

[0040] Figure 10 Figure 2 shows the organelle colocalization experiment of the fluorescent probe DHX-Fe of the present invention (A2780 cells). Scale bar: 20 μM. A is a cell image stained with Mito-Tracer; B is a cell image stained with DHX-Fe; C is a merged image of Mito-Tracer and DHX-Fe staining, showing colocalization within the cell; D is the grayscale value change of the two different trackers within a range of 0 to 60 pixels; and E is the fluorescence imaging of R Person. DETAILED DESCRIPTION

[0041] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0042] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0043] In the present invention, unless otherwise specified, all experimental raw materials used are commercially available products well known to those skilled in the art.

[0044] The A2780 cells and SKOV3 cells used in the present invention were purchased from Hunan Fenghui Biological Company; the mitochondrial dye Mito-Tracker was purchased from Beyotime Biotechnology Company.

[0045] Example 1

[0046] This embodiment provides a near-infrared ferrous ion fluorescent probe (DHX-Fe), and the specific preparation steps are as follows:

[0047] (1) Preparation of compound 1

[0048]

[0049] 5-Bromosalicylaldehyde (2.01 g, 10 mmol) and cesium carbonate (7.85 g, 24.12 mmol) were added to a reaction flask, which was then evacuated and filled with argon. 2-Bromocyclohex-1-ene-1-carbaldehyde (CAS-38127-47-8) (2.26 g, 2.06 mmol) was dissolved in DMF and added to the reaction flask. The reaction was stirred at 25°C for 12 h. After the reaction, the product was extracted with water and anhydrous dichloromethane. The organic layer was dried over anhydrous sodium sulfate, filtered, and the organic solvent was rotary evaporated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 20:1) to obtain 1.71 g of compound 1 as an orange-yellow solid in a yield of 45%.

[0050] The NMR data of compound 1 are: 1 H NMR (400 MHz, Chloroform- d )δ 10.24 (s, 1H),7.32-7.25 (m, 1H), 7.20 (d, J = 2.5 Hz, 1H), 6.90 (d, J = 8.6 Hz, 1H), 6.51 (d, J =1.3 Hz, 1H), 2.52 (ddd, J = 7.8, 5.8, 1.7 Hz, 2H), 2.37 (t, J = 6.0 Hz, 2H), 1.65(p, J = 6.2 Hz, 2H). 13 C NMR (101 MHz, Chloroform- d ) δ 187.95, 159.53, 150.90,132.39, 131.13, 128.91, 125.06, 122.84, 117.01, 115.92, 113.77, 30.03, 21.32,20.06. H RMS[M+H] + : C 14 H 11 BrO2, theoretical calculated value, 290.9942; actual value 291.0010.

[0051] The structural formula of compound 1 is: .

[0052] (2) Preparation of compound 2

[0053] The reaction formula is as follows:

[0054]

[0055] Compound 1 (580 mg, 2 mmol) obtained in step (1) and 1-ethyl-2-methylquinolinium iodide (CAS-606-55-3) (378.6 mg, 2.2 mmol) were added to a reaction flask, vacuumed, supplemented with argon, and then acetic anhydride (10 mL) was added as a solvent. The mixture was refluxed at 110 °C for 10 h. After the reaction, water was added and the mixture was filtered under reduced pressure using a Buchner funnel and dried to obtain 650 mg of black purple solid compound 2 with a yield of 48%.

[0056] The NMR data of compound 2 are: 1 H NMR (400 MHz, DMSO-d6) δ 8.82 (d, J = 9.1 Hz,1H), 8.60 (d, J = 9.1 Hz, 1H), 8.53 (d, J = 15.1 Hz, 1H), 8.44 (d, J = 9.0 Hz,1H), 8.30 (d, J = 7.3 Hz, 1H), 8.10 (t, J = 7.9 Hz, 1H), 7.86 (t, J = 7.5 Hz, 1H),7.63-7.54 (m, 2H), 7.40 (d, J = 8.7 Hz, 1H), 6.98 (s, 1H), 6.88 (d, J = 15.1 Hz,1H), 4.99 (q, J = 6.9 Hz, 2H), 2.67 (dt, J = 10.1, 6.0 Hz, 4H), 1.86 -1.78 (m,2H), 1.54 (t, J = 7.1 Hz, 3H). H RMS[M+H] + : C 26 H 23 BrNO + , theoretical calculated value, 444.0958; actual value, 444.0958.

[0057] The structural formula of compound 1 is: .

[0058] (3) Preparation of near-infrared ferrous ion fluorescent probe (DHX-Fe)

[0059]

[0060] Compound 2 (565 mg, 1.27 mmol) obtained in step (2), 4-amino-2,2,6,6-tetramethylpiperidinyl 1-oxyl radical (CAS-14691-88-4) (1.089 mg, 6.36 mmol), cesium carbonate (1.034 mg, 3.175 mmol), 2-dicyclohexylphosphino-2'-(N,N-dimethylamino)biphenyl (CAS-213697-53-1) (50 mg, 0.127 mmol) and trisdibenzylideneacetone dipalladium (CAS-51364-51-3) (116 mg, 0.127 mmol) were added to the reaction flask in sequence. After evacuation and nitrogen filling, 1, 4-dioxane was added and the reaction was carried out at 100°C for 6 h. After the reaction, the reaction solution was extracted with water and anhydrous dichloromethane, and the crude product was dried by rotary evaporation to obtain the crude product, which was purified by column chromatography (dichloromethane:methanol = 45:1), to obtain 380 mg of blue-green solid, namely the near-infrared ferrous ion fluorescent probe DHX-Fe of the present invention, with a yield of 56%.

[0061] The data of the near-infrared ferrous ion fluorescent probe DHX-Fe high-resolution mass spectrometry (HRMS) are shown in the attached Figure 1 shown.

[0062] The theoretical calculated value and the actual measured value of the molecular ion peak [M+H]+ are 535.3193 and 535.3188, respectively. According to the HRMS data, the molecular formula of DHX-Fe is: C 35 H 41 N3O2 + .

[0063] In order to further confirm the structure of the near-infrared ferrous ion fluorescent probe DHX-Fe of the present invention, DHX-Fe samples and DHX-Fe and ferrous ion (Fe) were prepared. 2+ ) combined with the sample (i.e. DHX-Fe + 10eq Fe 2+ ) Use electron spin resonance (ESR) or other techniques that can detect the presence of free radicals to test the DHX-Fe sample, record and organize the obtained data, and generate an ERP spectrum. For details, see the attached Figure 2 shown.

[0064] By the attached Figure 2 The data show that the ERP diagram of the DHX-Fe sample contains three EPR signal peaks, including a 1:1:1 three-line EPR signal of a typical nitroxide free radical, indicating that the DHX-Fe structure contains nitroxide free radicals. The presence of nitroxide free radicals gives DHX-Fe special fluorescence properties and reaction activity, which is crucial for its application as a fluorescent probe.

[0065] In summary, through the analysis of ERP graphs and HRMS data, it was confirmed that the DHX-Fe structure contained nitroxide radicals, and its molecular formula was determined to be C 35 H 41 N3O2 + These results provide strong support for the application of DHX-Fe as a near-infrared fluorescent probe for ferrous ions.

[0066] Example 2

[0067] This example, based on Example 1, measures electromagnetic paramagnetic resonance (EPR) of the fluorescent probe DHX-Fe before and after the response to ferrous ions.

[0068] Take 50 μL DHX-Fe (5 μM ethanol solution) to test the NO • signal of DHX-Fe by EPR, and record the spectrum at room temperature as the control group; add an equal amount of ferrous ions 10 eq Fe 2+ , ensuring that the molar ratio of ferrous ions to probe molecules is 10:1. After 10 min of reaction, the mixed solution is subjected to EPR detection again and the EPR spectrum is recorded as the experimental group. For specific results, see the attached Figure 3 shown.

[0069] By the attached Figure 3 The data shows that the original DHX-Fe solution shows a strong EPR signal of the typical 1:1:1 three-line of TEMPOL, which indicates that DHX-Fe itself has significant NO• free radical characteristics; when ferrous ions are added to the DHX-Fe solution and reacted for 10 minutes, the EPR signal of the solution is significantly weakened, indicating that a rapid interaction occurs between DHX-Fe and ferrous ions, resulting in a decrease in the number of NO• free radicals or a change in their properties.

[0070] By measuring the EPR changes in the fluorescent probe DHX-Fe before and after its response to ferrous ions, the interaction mechanism between the two was successfully revealed. This discovery not only provides strong evidence for the ferrous ion detection capability of the DHX-Fe probe, but also provides a useful reference for further exploring its application range and detection sensitivity.

[0071] Example 3

[0072] In this example, based on Example 1, the ultraviolet absorption spectra of the near-infrared ferrous ion fluorescent probe DHX-Fe of the present invention before and after responding to ferrous ions were measured.

[0073] Accurately weigh the probe DHX-Fe, dissolve it in DMSO, and prepare a probe stock solution with a concentration of 1.0 mM. Store it at 4-8°C until ready for use. Prepare a 10 mM ferrous ion standard solution with oxygen-free distilled water. Add 25 μL of the DHX-Fe probe stock solution (final concentration 5 μM) to a 5 mL volumetric flask, add the solvent (EtOH:PBS = 3:7) and adjust the volume. Then, add 10 eq (25 μL) of ferrous ion. After reacting for 10 minutes, scan the solution using a UV spectrometer and record its UV absorption spectrum. For specific results, see the attached figure. Figure 4 shown.

[0074] By the attached Figure 4 The data show that when only the DHX-Fe probe is present, the ultraviolet absorption spectrum shows two maximum absorption peaks at 600 nm and 660 nm; when ferrous ions are added to the probe solution and reacted for 10 minutes, the absorption peaks of the probe at 600 nm and 660 nm increase significantly, indicating that new substances are formed after the DHX-Fe probe reacts with ferrous ions, thereby affecting its absorption characteristics at 600 nm and 660 nm. This also verifies the effectiveness of the DHX-Fe probe as a near-infrared ferrous ion fluorescent probe, that is, it can respond to the presence of ferrous ions and undergo detectable spectral changes.

[0075] Example 4

[0076] This example, based on Example 1, measures the ultraviolet emission spectrum of the fluorescent probe DHX-Fe before and after the response to ferrous ions.

[0077] Ferrous ion solutions of different concentrations (0 μM to 50 μM) were gradually added to the DHX-Fe (5 μM, ethanol:PBS = 3:7) probe solution. After 20 min of reaction, the UV emission spectra of the mixed solutions were measured. For specific results, see the attached Figure 5 shown.

[0078] By the attached Figure 5 The data show that the fluorescence emission spectra of the probe DHX-Fe before and after responding to ferrous ions show that the emission intensity of the mixed solution gradually increases with the increase of ferrous ion concentration, which indicates that the probe DHX-Fe has good concentration response characteristics to ferrous ions; when the ferrous ion concentration is low, the change in fluorescence intensity is more significant, showing the high sensitivity of the probe.

[0079] Example 5

[0080] Based on Example 1, this example measures the response time dependence of the fluorescent probe DHX-Fe on ferrous ions and studies the change in fluorescence intensity before and after the reaction of the probe DHX-Fe with ferrous ions.

[0081] Add 50 μM ferrous ion solution to the DHX-Fe (5 μM, ethanol:PBS=3:7) probe, and then measure the fluorescence intensity of the mixed solution over time (Ex / Em: 610nm / 690nm, slit bandwidth: 3nm / 3nm). Record the fluorescence intensity at different time points (over time). For specific measurement results, see the attached Figure 6 shown.

[0082] By the attached Figure 6 The data shows that the addition of ferrous ions to the DHX-Fe probe solution produces a rapid fluorescence signal response, indicating a rapid interaction between the probe and the ferrous ions. Over time, the fluorescence intensity gradually approaches saturation, reaching a maximum and equilibrium after 5 minutes of detection. Over the same time period, the fluorescence intensity remains unchanged when no ferrous ions are added, confirming that the increase in fluorescence intensity is due to the interaction between the probe and the ferrous ions.

[0083] This example successfully revealed the response time dependence of the fluorescent probe DHX-Fe by measuring the change in fluorescence intensity before and after the reaction with ferrous ions. The experimental results show that the DHX-Fe probe can rapidly respond to the presence of ferrous ions and reach the maximum fluorescence intensity within a short period of time, indicating that the probe has a fast response characteristic and is suitable for real-time monitoring of changes in ferrous ions. This provides strong support for the application of the DHX-Fe probe in ferrous ion detection.

[0084] Example 6

[0085] This example, based on Example 1, determines the selectivity and interference of the fluorescent probe DHX-Fe in detecting ferrous ions.

[0086] To test the selectivity of the fluorescent probe for ferrous ions, the responses of the fluorescent probe to different analytes were explored. After adding other analytes to the probe solution, the changes in the fluorescence signal were observed.

[0087] Accurately weigh a certain amount of the following metal salts: NaCl, KCl, LiNO₃, MgCl₂, CaCl₂, FeCl₃•6H₂O, HgCl₂, CrCl₃•6H₂O, CoSO₄, MnSO₄, AgNO₃, CdCl₂•H₂O, NiCl₂•6H₂O, Cu(NO₃)₂•3H₂O, ZnCl₂, AlNO₃•9H₂O, [Cu(CH₃CN)₄][PF₆], dissolve in distilled water, and dilute to 10 mL. Mix thoroughly by sonication to prepare a 10 mM analyte standard solution. Store at 4-8°C until ready for use. Add 25 μL of the DHX-Fe probe stock solution (final concentration 5 μM) to a 5 mL volumetric flask, add solvent (EtOH:PBS = 3:7) and dilute to volume. Then, add 5 eq of the interfering analyte and incubate the mixture at room temperature for 20 min before fluorescence measurement. Take DHX-Fe probe solution (5 μM) and add interfering analyte (5 eq) and Fe 2+ (10eq), the final volume of the analyte is controlled to be less than 1% of the total volume. The test conditions are: ex =610 nm, λ em =690 nm, silt band: 3nm / 3nm. For details, please refer to the attached Figure 7 shown.

[0088] By the attached Figure 7 The data show that even with other analytes (Fe 2+ 、Na + 、Cd 2+ 、Ag + 、Zn 2+ Cr 3+ 、Fe 3+ 、Hg 2+ 、Li + Mg 2+ 、Ni 2+ 、Cu 2+ 、Cu + , Ca 2+ 、Co 2+ 、Mn 2+ 、Al 3+ and K +) in the presence of, no particularly obvious fluorescence enhancement signal is observed, indicating that these analytes have less influence on the fluorescence intensity of the fluorescent probe, that is, the fluorescent probe does not have a significant response to these analytes; after adding ferrous ion, obvious fluorescence enhancement signal can still be observed at 690 nm, proving that the fluorescent probe DHX-Fe of the present invention has a high selectivity for ferrous ion, while the probe is tested together with other analytes and ferrous ion for the anti-interference ability of the probe to ferrous ion, when other analytes coexist with ferrous ion, the probe will not be affected by other analytes, and the detection of ferrous ion can still be achieved, indicating that the fluorescent probe DHX-Fe can still achieve accurate detection of ferrous ion in a complex environment, and has good anti-interference ability. The experimental results of the present embodiment show that the fluorescent probe DHX-Fe has a high selectivity and good anti-interference ability for ferrous ion, and is suitable for the detection of ferrous ion.

[0089] Example 7

[0090] In this example, based on Example 1, a laser confocal microscopy experiment was conducted to investigate the recognition of exogenous ferrous ions by the fluorescent probe DHX-Fe in human ovarian cancer A2780 cells.

[0091] A2780 cells were seeded in confocal microplates. In the DHX-Fe group, A2780 cells were incubated with 5 μM probe for 30 min. 2+ Group: Fe was firstly used at molar concentrations of 25 μM and 50 μM 2+ A2780 cells were incubated with exogenous ferrous ions for 1 h and washed with PBS to remove unbound Fe 2+ After that, the cells were incubated with 5 μM DHX-Fe probe (in HBSS) for 15 min. After treatment, the FV3000 laser confocal imaging (Ex / Em: 640nm / 680-720nm) was performed directly in each group. The test results are shown in the attached Figure 8 shown.

[0092] By the attached Figure 8 The data showed that compared with the DHX-Fe group, Fe 2+ The red channel fluorescence intensity of the group increased significantly. When exogenous ferrous ions were present in the cells, the DHX-Fe probe was able to bind to them and produce a stronger fluorescence signal. This probe can realize rapid and real-time detection of ferrous ions in cells.

[0093] Example 8

[0094] In this example, based on Example 1, a laser confocal microscopy experiment was conducted to investigate the recognition of endogenous ferrous ions by the fluorescent probe DHX-Fe in human ovarian cancer SKOV3 cells.

[0095] SKOV3 cells were seeded in confocal microplates. The DHX-Fe group served as a control group and was incubated with the probe DHX-Fe at a molar concentration of 5 μM for 30 min to observe the basal level of ferrous ions in uninduced cells. The Erastin group was incubated with the ferroptosis inducer Erastin at a molar concentration of 10 μM for 2 h and 4 h, respectively, and then incubated with the DHX-Fe probe for 30 min. The Erastin+Fer-1 group was simultaneously added with Erastin (10 μM) and Fer-1 (2 μM) and incubated with the DHX-Fe probe for 4 h, and then incubated with the DHX-Fe probe for 30 min. After treatment, cells in each group were treated with Hoechst 33342 (5 μg / mL, nuclear stain) and incubated for 15 min. The cells were then washed three times with PBS and directly imaged with FV3000 laser confocal microscopy (Hoechst 33342, Ex / Em: 405 nm / 435-475 nm; DHX-Fe, Ex / Em: 640 nm / 680-720 nm). For specific test results, see the attached Figure 9 shown.

[0096] By the attached Figure 9 The data show that in the DHX-Fe group, the fluorescence intensity of the red channel (DHX-Fe probe) was weak, indicating that the content of endogenous ferrous ions in uninduced SKOV3 cells was low; in the Erastin group, 2-4 hours after the addition of the ferroptosis inducer, the red channel fluorescence signal gradually increased, indicating that Erastin successfully induced cellular ferroptosis, resulting in an increase in the intracellular ferrous ion content, which was then captured by the DHX-Fe probe and produced a stronger fluorescence signal; in the Erastin+Fer-1 group, the red channel fluorescence signal intensity was lower than that of the group with only Erastin added. Fer-1 successfully inhibited Erastin-induced ferroptosis, verifying the specificity of the DHX-Fe probe in detecting ferrous ions elevated in ferroptosis-induced cells. The results showed that the DHX-Fe probe can be used to detect the increase of ferrous ions in cells induced by ferroptosis in real time; Erastin, as a ferroptosis inducer, can significantly increase the content of ferrous ions in cells and is effectively captured by the DHX-Fe probe; Fer-1, as a ferroptosis inhibitor, its addition may reduce or inhibit the increase of ferrous ions induced by Erastin, thereby further verifying the specificity of the probe. This experiment successfully verified the application potential of the DHX-Fe probe in detecting the increase of ferrous ions in cells induced by ferroptosis through laser confocal imaging technology.

[0097] Example 10

[0098] This example, based on Example 1, conducts an organelle co-localization experiment of the fluorescent probe DHX-Fe in human ovarian cancer A2780 cells.

[0099] A2780 cells were seeded in a confocal microplate and allowed to adhere. A commercial mitochondrial dye, Mito-Tracer (100 nM), was added to the culture medium and incubated for 30 min. The cells were then washed three times with PBS. DHX-Fe (5 μM) probe was then added and incubated for 30 min. The cells were then washed three times with PBS before direct FV3000 laser confocal imaging (Mito-Tracker, Ex / Em: 488 nm / 510-530 nm; DHX-Fe: Ex / Em: 640 nm / 680-720 nm). For details, see the attached figure. Figure 10 shown.

[0100] By the attached Figure 10 The data shows that Figure 10 In the figure, A shows a cell image stained with Mito-Tracer; B shows a cell image stained with DHX-Fe; C shows the colocalization of Mito-Tracer and DHX-Fe within the cell; D shows the grayscale change of the two tracers within a region ranging from 0 to 60 pixels; and E shows the fluorescence image of R Person. When A2780 cells were co-incubated with the mitochondrial dye and the probe DHX-Fe, fluorescence signals were generated in corresponding channels without cross-interference. The Pearson coefficient R of 0.91 was calculated by superimposing the fluorescence signals of the DHX-Fe red channel and the mitochondrial green channel, indicating a high degree of colocalization between DHX-Fe and the mitochondrial dye Mito-Tracer. This further confirms that DHX-Fe can be localized to mitochondria and can be used to image ferrous ions in mitochondrial subcellular organelles in human ovarian cancer A2780 cells. This provides an important tool for understanding the role of ferrous ions in cellular metabolism and disease progression.

[0101] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A near-infrared ferrous ion fluorescent probe, characterized in that: The structure of the fluorescent probe is as follows: 。 2. The method for preparing the near-infrared ferrous ion fluorescent probe according to claim 1, wherein include: S1, under argon protection, 5-bromosalicylaldehyde was mixed with cesium carbonate, dissolved and reacted with 2-bromocyclohex-1-ene-1-carbaldehyde, extracted, the organic layer was dried, and purified by column chromatography to prepare compound 1; The structural formula of the compound 1 is: ; S2, under argon protection, compound 1 in S1 was mixed with 1-ethyl-2-methylquinolinium iodide, a solvent was added for reaction, the mixture was filtered under reduced pressure, and the mixture was dried to obtain compound 2; The structural formula of the compound 2 is: ; S3, under nitrogen protection, the compound 2 obtained in S2, 4-amino-2,2,6,6-tetramethylpiperidinyl 1-oxyl free radical, cesium carbonate, 2-dicyclohexylphosphino-2'-(N,N-dimethylamino)biphenyl and trisdibenzylideneacetone dipalladium were mixed for reaction, extracted, dried, and purified by column chromatography to obtain a near-infrared ferrous ion fluorescent probe.

3. The method for preparing the near-infrared ferrous ion fluorescent probe according to claim 2, wherein In S1, the molar ratio of 5-bromosalicylaldehyde, cesium carbonate, and 2-bromocyclohex-1-ene-1-carbaldehyde is 4-5:10-12:1, the reaction temperature is 24°C-26°C, and the reaction time is 10-14 h; the solvent used for the extraction is water and dichloromethane, and the mobile phase used for the column chromatography purification is petroleum ether and ethyl acetate in a volume ratio of 20:

1.

4. The method for preparing the near-infrared ferrous ion fluorescent probe according to claim 2, wherein In S2, the molar ratio of the compound 1 to 1-ethyl-2-methylquinolinium iodide is 1:1-2, the solvent is acetic anhydride, the reaction temperature is 100°C-120°C, and the reaction time is 8 h-12 h.

5. The method for preparing the near-infrared ferrous ion fluorescent probe according to claim 2, wherein In S3, the molar ratio of the compound 2, 4-amino-2,2,6,6-tetramethylpiperidinyl 1-oxyl free radical, cesium carbonate, 2-dicyclohexylphosphino-2'-(N,N-dimethylamino)biphenyl and trisdibenzylideneacetone dipalladium is 1:5~6:2~3:0.1~0.2:0.1~0.2, the reaction temperature is 90°C~110°C, and the reaction time is 5 h~7 h.

6. The method for preparing the near-infrared ferrous ion fluorescent probe according to claim 2, wherein: In S3, the extraction uses water and dichloromethane as solvents, and the column chromatography purification uses dichloromethane and methanol in a volume ratio of 45:1 as mobile phases.

7. The near-infrared ferrous ion fluorescent probe according to claim 1 is used in the preparation of a reagent for measuring, screening or detecting ferrous ions, characterized in that: The ferrous ions are endogenous or exogenous ferrous ions in cells.

8. Use of the near-infrared ferrous ion fluorescent probe according to claim 1 in cell fluorescence imaging for non-disease diagnosis and treatment purposes.

9. A kit for detecting the presence of ferrous ions in a sample or measuring the ferrous ion content in a sample, characterized in that: The kit comprises the near-infrared ferrous ion fluorescent probe according to claim 1.

10. A method for detecting the presence of ferrous ions in a sample or determining the ferrous ion content in a sample, characterized in that: include: (1) Using the kit described in claim 9, a near-infrared ferrous ion fluorescent probe is brought into contact with a sample to form a fluorescent compound; (2) Determine the fluorescence properties of the fluorescent compound.

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