Near-infrared ferrous ion fluorescent probe as well as preparation and application thereof

Abstract

The invention discloses a near-infrared iron ion fluorescent probe as well as preparation and application thereof, and belongs to the technical field of analytical chemistry and biology. The excitation wavelength and the emission wavelength of the fluorescent probe disclosed by the invention are both located in a near-infrared region, the fluorescent probe has excellent selection performance on ferrous ions, can realize rapid detection on endogenous and exogenous ferrous ions of cells, can effectively avoid interference of autofluorescence of a sample, and improves the signal-to-noise ratio; the probe can be positioned in mitochondria, is crucial to research on the dynamic state and function of ferrous ions in the mitochondria, can rapidly image endogenous and exogenous ferrous ions in tumor cells in real time, shows a good biological fluorescence imaging application prospect, provides a powerful tool for tumor research, is beneficial to revealing the effect of the ferrous ions in tumors, and has a wide application prospect. The targeted treatment effect is evaluated, and the treatment scheme is optimized. The kit has potential clinical value in the aspects of tumor diagnosis, treatment monitoring, prognosis evaluation and the like, and has important technical effects and wide application prospects in biomedical research and clinical diagnosis.

Description

Technical Field

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

[0002] As an essential trace element for the human body, iron plays an indispensable role in the body. Its redox activity is the key to many biological processes (such as oxygen transport, electron transfer and enzyme catalysis). 2+ ) concentration imbalance, especially in organelles such as mitochondria and lysosomes, is closely related to the occurrence and development of various diseases such as neurodegenerative diseases and cancer. When the intracellular iron homeostasis is unbalanced, it will lead to the release of harmful reactive oxygen species (ROS), which will in turn cause oxidative stress and cell damage. This imbalance is closely related to the occurrence and development of various diseases, including neurodegenerative diseases, cancer, etc.

[0003] In order to accurately monitor the concentration of ferrous ions in cells, 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), have high accuracy but are complex to operate and require expensive equipment, which limits 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 simple operation, low cost, and intuitive detection results. Fluorescent probes specifically bind to ferrous ions to produce obvious changes in fluorescence signals, thereby achieving high sensitivity and high selectivity detection of ferrous ions. Although existing fluorescent probes have made certain progress in detecting ferrous ions, there are still some problems, such as insufficient selectivity and the need to improve sensitivity. Therefore, the development of a ferrous ion fluorescent probe with high selectivity, high sensitivity, and real-time imaging in vivo is of great significance for in-depth understanding of the biological function 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 view of the problems that the existing ferrous ion fluorescent probes have low selectivity and need to improve their sensitivity 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: The present invention provides a near-infrared ferrous ion fluorescent probe, the structure of the fluorescent probe is as follows: .

[0007] The present invention provides a method for preparing the above-mentioned near-infrared ferrous ion fluorescent probe, comprising: S1, under argon protection, 5-bromosalicylaldehyde and cesium carbonate were mixed, dissolved and reacted with 2-bromocyclohex-1-ene-1-carboxaldehyde, 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 to react, 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 radical, cesium carbonate, 2-dicyclohexylphosphino-2'-(N,N-dimethylamino)biphenyl and tris(dibenzylideneacetone)dipalladium were mixed for reaction, extracted, dried, and purified by column chromatography to obtain a near-infrared ferrous ion fluorescent probe; .

[0008] In S1, the molar ratio of 5-bromosalicylaldehyde, cesium carbonate and 2-bromocyclohex-1-ene-1-carboxaldehyde 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.

[0009] 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.

[0010] In S3, the molar ratio of the compound 2, 4-amino-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, cesium carbonate, 2-dicyclohexylphosphino-2'-(N,N-dimethylamino)biphenyl and tridibenzylideneacetone 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.

[0011] 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.

[0012] The present invention provides the use of the near-infrared ferrous ion fluorescent probe in the preparation of a reagent for measuring, screening or detecting ferrous ions, wherein the ferrous ions are endogenous or exogenous ferrous ions in cells.

[0013] The present invention provides application of the near-infrared ferrous ion fluorescent probe in cell fluorescence imaging.

[0014] 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: (1) using the above kit to contact the near-infrared ferrous ion fluorescent probe with a sample to form a fluorescent compound; (2) Determining the fluorescence properties of the fluorescent compound.

[0015] Compared with the prior art, the present invention achieves the following technical effects: The near-infrared ferrous ion fluorescent probe provided by the present invention has excitation and emission wavelengths both in the near-infrared region, and this feature enables the probe to have stronger penetration ability in biological tissues, reduces light scattering and background noise, and improves the clarity and depth of imaging; 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 the interference of the excitation light and improve the signal-to-noise ratio of the signal; can be located in mitochondria, which are the main energy producers in cells and important sites for ferrous ion metabolism, and are of great significance for studying the dynamics and functions of ferrous ions in mitochondria; can be used together with other commercial dyes without generating signal interference, which increases its application flexibility in complex biological environments; can realize 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.

[0016] 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 easy to obtain, the cost is low, and it is suitable for large-scale production, which is conducive to the practical application and promotion of the probe.

[0017] 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, and provides a powerful tool for the research of tumor growth, metastasis and treatment. Through imaging, the molecular mechanism of tumor growth, metastasis and treatment can be deeply understood, which is helpful to reveal the role of ferrous ions in tumor development, 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, and also has important technical effects and broad application prospects in biomedical research and clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the high resolution mass spectrometry (HRMS) spectrum of the probe DHX-Fe of the present invention; Figure 2 is the electron paramagnetic resonance (EPR) spectrum of the probe DHX-Fe of the present invention; Figure 3 The electron paramagnetic resonance (EPR) spectrum of the probe DHX-Fe of the present invention before and after the response to ferrous ions; Figure 4 The UV absorption spectrum of the probe DHX-Fe of the present invention before and after responding to ferrous ions (0-10eq); Figure 5 Fluorescence emission spectra of the probe DHX-Fe of the present invention before and after responding to (0-10eq) ferrous ions; Figure 6 Fluorescence intensity changes of the probe DHX-Fe of the present invention at different times after adding ferrous ions (Ex / Em: 610nm / 690nm, slitband: 3nm / 3nm); Figure 7 The fluorescence spectrum of the probe DHX-Fe of the present invention for the selectivity and interference of ferrous ions; 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; Fig. 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; Fig.10This is an organelle co-localization experiment of the fluorescent probe DHX-Fe of the present invention (A2780 cells), scale 20μM; A is a cell image stained with Mito-Tracer; B is a cell image stained with DHX-Fe dye; C is a merged staining image of Mito-Tracer and DHX-Fe, showing the co-localization in the cell, D is the change in grayscale value of two different trackers in the area with a distance of 0 to 60 pixels, and E is the fluorescence imaging of R Person. DETAILED DESCRIPTION

[0019] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme 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 described embodiments 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 creative work should fall within the scope of protection of the present invention.

[0020] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0021] In the present invention, unless otherwise specified, all experimental raw materials used are commercially available products well known to those skilled in the art.

[0022] 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.

[0023] Example 1 This embodiment provides a near-infrared ferrous ion fluorescent probe (DHX-Fe), and the specific preparation steps are as follows: (1) Preparation of compound 1

[0024] 5-Bromosalicylaldehyde (2.01 g, 10 mmol) and cesium carbonate (7.85 g, 24.12 mmol) were added to the reaction flask, and the mixture was evacuated and supplemented with argon. 2-Bromocyclohex-1-ene-1-carboxaldehyde (CAS-38127-47-8) (2.26 g, 2.06 mmol) was dissolved in DMF and added to the reaction flask. The mixture was stirred at 25 °C for 12 h. After the reaction, the mixture was extracted with water and anhydrous dichloromethane, and 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 orange-yellow solid compound 1 with a yield of 45%.

[0025] 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.

[0026] The structural formula of compound 1 is: .

[0027] (2) Preparation of Compound 2 The reaction formula is as follows:

[0028] 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, vacuumized, 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 dark purple solid compound 2 with a yield of 48%.

[0029] 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.

[0030] The structural formula of compound 1 is: .

[0031] (3) Preparation of near-infrared ferrous ion fluorescent probe (DHX-Fe)

[0032] 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 tris(dibenzylideneacetone)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%.

[0033] The near-infrared ferrous ion fluorescent probe DHX-Fe high-resolution mass spectrometry (HRMS) data are shown in the attached Figure 1 shown.

[0034] The theoretical calculated value and the actual measured value of the molecular ion peak [M+H]+ are 535.0958 and 535.3188, respectively. According to the HRMS data, the molecular formula of DHX-Fe is: C 26 H 23 BrNO + .

[0035] 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.

[0036] By the attached Figure 2 From the data, we can see 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.

[0037] 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 26 H 23 BrNO + These results provide strong support for the application of DHX-Fe as a near-infrared ferrous ion fluorescent probe.

[0038] Example 2 Based on Example 1, this example measures the electromagnetic paramagnetic resonance (EPR) of the fluorescent probe DHX-Fe before and after the response to ferrous ions.

[0039] 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.

[0040] By the attached Figure 3 The data show that the original DHX-Fe solution shows a strong 1:1:1 three-line EPR signal typical 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.

[0041] By measuring the EPR changes of the fluorescent probe DHX-Fe before and after the 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 ability of the DHX-Fe probe, but also provides a useful reference for further exploring its application range and detection sensitivity.

[0042] Example 3 Based on Example 1, this example measures the ultraviolet absorption spectrum of the near-infrared ferrous ion fluorescent probe DHX-Fe of the present invention before and after responding to ferrous ions.

[0043] Weigh the probe DHX-Fe accurately, dissolve it in DMSO to prepare a probe stock solution with a concentration of 1.0 mM, and store it at 4-8 °C for later use; prepare a ferrous ion standard solution with a molar concentration of 10 mM with oxygen-free distilled water; add 25 μL of DHX-Fe probe stock solution (final concentration 5 μM) to a 5 mL volumetric flask, add solvent (EtOH: PBS = 3:7) and make up to volume, then add 10 eq (25 μL) of ferrous ions, react for 10 minutes, scan the solution after the reaction using a UV spectrometer, and record its UV absorption spectrum. For specific results, see the attached Figure 4 shown.

[0044] 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.

[0045] Example 4 This example is based on Example 1, and measures the ultraviolet emission spectrum of the fluorescent probe DHX-Fe before and after the response to ferrous ions.

[0046] Different concentrations (0 μM~50 μM) of ferrous ion solution 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.

[0047] By the attached Figure 5 From the data, we can see that the fluorescence emission spectrum of the probe DHX-Fe before and after responding to ferrous ions shows 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.

[0048] Example 5 Based on Example 1, this example determines the response time dependence of the fluorescent probe DHX-Fe and ferrous ions, and studies the change in fluorescence intensity before and after the reaction of the probe DHX-Fe and ferrous ions.

[0049] 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), and record the fluorescence intensity at different time points (over time). For specific measurement results, see the attached Figure 6 shown.

[0050] By the attached Figure 6 The data show that after adding ferrous ion solution to the DHX-Fe probe solution, a rapid fluorescence signal response can be generated, indicating that a rapid interaction occurs between the probe and the ferrous ions; as time goes on, the fluorescence intensity gradually tends to saturation, and when the detection time is 5 minutes, the fluorescence intensity reaches the maximum value and reaches equilibrium. In the same period of time, when no ferrous ions are added, the fluorescence intensity does not change significantly, confirming that the increase in fluorescence intensity is due to the interaction between the probe and the ferrous ions.

[0051] 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 quickly respond to the presence of ferrous ions and reach the maximum fluorescence intensity in a short 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.

[0052] Example 6 Based on Example 1, this example determines the selectivity and interference of the fluorescent probe DHX-Fe in detecting ferrous ions.

[0053] In order to test the selectivity of the fluorescent probe for ferrous ions, the responses of the fluorescent probe to different analytes were investigated. After adding other analytes to the probe solution, the changes in the fluorescence signal were observed.

[0054] 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 make up to 10 mL, mix by ultrasonication to make a 10 mM analyte standard solution, and store at 4-8 °C for later use. Add 25 μL of DHX-Fe probe mother solution (final concentration 5 μM) to a 5 mL volumetric flask, add solvent (EtOH: PBS = 3:7) and make up to volume, then add 5 eq of interfering analyte and place the mixed solution at room temperature for 20 min before fluorescence measurement. Take the DHX-Fe probe solution (5 μM) and add the 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. Test conditions are: ex =610 nm, λ em =690 nm, silt band: 3nm / 3nm. For details, please refer to the attached Figure 7 shown.

[0055] By the attached Figure 7 The data show that even in 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 little effect 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 ions, obvious fluorescence enhancement signals can still be observed at 690 nm, proving that the fluorescent probe DHX-Fe of the present invention has a high degree of selectivity for ferrous ions, and the probe is tested together with other analytes and ferrous ions for the anti-interference ability of the probe to ferrous ions. When other analytes coexist with ferrous ions, the probe will not be affected by other analytes, and the detection of ferrous ions can still be achieved, indicating that the fluorescent probe DHX-Fe can still achieve accurate detection of ferrous ions 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 degree of selectivity and good anti-interference ability for ferrous ions, and is suitable for the detection of ferrous ions.

[0056] Example 7 Based on Example 1, this example conducted a laser confocal microscopy experiment on the recognition of exogenous ferrous ions by the fluorescent probe DHX-Fe in human ovarian cancer A2780 cells.

[0057] A2780 cells were seeded in confocal microplates. DHX-Fe group: A2780 cells were incubated with 5 μM probe for 30 min; Fe 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 then 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 each treatment, FV3000 laser confocal imaging (Ex / Em: 640nm / 680-720nm) was performed directly. The test results are shown in the attached Figure 8 shown.

[0058] By the attached Figure 8 The data showed that compared with the DHX-Fe group, Fe 2+ The fluorescence intensity of the red channel of the group was significantly increased. When exogenous ferrous ions were present in the cells, the DHX-Fe probe could bind to them and produce a stronger fluorescence signal. The probe could realize rapid and real-time detection of ferrous ions in cells.

[0059] Example 8 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.

[0060] SKOV3 cells were seeded in confocal microplates. DHX-Fe group: as the control group, cells were incubated with 5 μM probe DHX-Fe for 30 min to observe the basal level of ferrous ions in uninduced cells. Erastin group: cells were incubated with 10 μM ferroptosis inducer Erastin for 2 h and 4 h, respectively, and then incubated with DHX-Fe probe for 30 min. Erastin+Fer-1 group: cells were incubated with Erastin (10 μM) and Fer-1 (2 μM) for 4 h, and then incubated with DHX-Fe probe for 30 min. After treatment, each group was added with Hoechst33342 (5 μg / mL, nuclear stain) and incubated for 15 min, then washed 3 times with PBS, and directly performed FV3000 laser confocal imaging (Hoechst33342, Ex / Em: 405nm / 435-475nm; DHX-Fe, Ex / Em: 640nm / 680-720nm). For specific test results, see the attached Fig. 9 shown.

[0061] By the attached Fig. 9 The data show that, DHX-Fe group: the fluorescence intensity of the red channel (DHX-Fe probe) is weak, indicating that the content of endogenous ferrous ions in uninduced SKOV3 cells is low; Erastin group: 2-4 hours after the addition of ferroptosis inducer, the red channel fluorescence signal gradually increased, indicating that Erastin successfully induced cell 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; Erastin+Fer-1 group: the red channel fluorescence signal intensity was lower than that of the group with only Erastin added, and 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 in real time the elevated ferrous ions in cells induced by ferroptosis; Erastin, as an inducer of ferroptosis, can significantly increase the content of ferrous ions in cells and be effectively captured by the DHX-Fe probe; Fer-1, as an inhibitor of ferroptosis, 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 elevated ferrous ions in cells induced by ferroptosis through laser confocal imaging technology.

[0062] Example 10 This example is based on Example 1, and an experiment on the co-localization of the fluorescent probe DHX-Fe in organelles of human ovarian cancer A2780 cells is carried out.

[0063] A2780 cells were seeded in a confocal dish, and after the cells adhered to the wall, commercial mitochondrial dye Mito-Tracer (100nM) was added to the culture medium, incubated for 30 min, washed 3 times with PBS, and then DHX-Fe (5μM) probe was added to incubate the cells for 30 min, washed 3 times with PBS, and then directly performed FV3000 laser confocal imaging (Mito-Tracker, Ex / Em: 488nm / 510-530nm; DHX-Fe: Ex / Em: 640nm / 680-720nm). For details, see the attached Fig.10 shown.

[0064] By the attached Fig.10 The data shows that Fig.10 In the figure, A is a cell image stained with Mito-Tracer; B is a cell image stained with DHX-Fe dye; C is a merged image of Mito-Tracer and DHX-Fe staining, showing the co-localization in the cell; D is the change in grayscale value of two different tracers in the area with a distance of 0 to 60 pixels; and E is the fluorescence imaging of R Person. After A2780 cells were co-incubated with mitochondrial dyes and probe DHX-Fe dyes, fluorescence signals were generated in the corresponding channels without mutual interference. By superimposing the fluorescence signals of the probe DHX-Fe red channel and the mitochondrial green dye channel, the Pearson coefficient R=0.91 was calculated, indicating that there was a high degree of co-localization between DHX-Fe and the mitochondrial dye Mito-Tracer, further confirming that DHX-Fe can be localized in mitochondria, and the probe DHX-Fe can be localized in mitochondrial subcellular organelles in human ovarian cancer A2780 cells, and can be used for imaging detection of ferrous ions in organelles. This provides an important tool for understanding the role of ferrous ions in cell metabolism and disease development.

[0065] The above contents are 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, characterized in that: include: S1, under argon protection, 5-bromosalicylaldehyde and cesium carbonate were mixed, dissolved and reacted with 2-bromocyclohex-1-ene-1-carboxaldehyde, 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 to react, 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 radical, cesium carbonate, 2-dicyclohexylphosphino-2'-(N,N-dimethylamino)biphenyl and tris(dibenzylideneacetone)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, characterized in that: In S1, the molar ratio of 5-bromosalicylaldehyde, cesium carbonate and 2-bromocyclohex-1-ene-1-carboxaldehyde 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, characterized in that: 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, characterized in that: In S3, the molar ratio of the compound 2, 4-amino-2,2,6,6-tetramethylpiperidine 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, characterized in that: 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.

9. A kit for detecting the presence of ferrous ions in a sample or determining the ferrous ion content in a sample, characterized in that: The kit comprises the near-infrared ferrous ion fluorescent probe described in 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 to contact a near-infrared ferrous ion fluorescent probe with a sample to form a fluorescent compound; (2) Determining the fluorescence properties of the fluorescent compound.

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