CN113651759A9

CN113651759A9 - Detection of Fe in organism2+Two-photon fluorescent probe and preparation method and application thereof

Info

Publication number: CN113651759A9
Application number: CN202110850514.6A
Authority: CN
Inventors: 张承武; 陆瑶; 李林; 吴越; 徐家佳; 王延宾; 黄维
Original assignee: Nanjing Tech University
Current assignee: Nanjing Tech University
Priority date: 2021-07-27
Filing date: 2021-07-27
Publication date: 2021-12-24
Anticipated expiration: 2041-07-27
Also published as: CN113651759B; CN113651759A

Abstract

The invention relates to a pyrimidine-based dye which can be used for quickly and sensitively detecting Fe in organisms through light regulation²⁺The two-photon fluorescent probe belongs to the field of organic fluorescent probes. The fluorescent probe is a PyFe fluorescent probe, and the structural formula of the PyFe fluorescent probe is shown as a formula (I). The fluorescent probe can accurately detect Fe in organisms²⁺And the interference of other ionic amino acids in the cells is avoided. The probe has extremely high response rate and high ion selectivity, can realize in-vitro naked eye detection and also has good biocompatibility. Fluorescence confocal imaging experiments show that the probe has good cell permeability, has no toxic or side effect on cells and organisms, has two-photon property, and can realize the depth of living animals or tissuesAnd (6) imaging.

Description

Detection of Fe in organism2+Two-photon fluorescent probe and preparation method and application thereof

Technical Field

The invention relates to a pyrimidine-based dye which can be used for quickly and sensitively detecting Fe in organisms through light regulation²⁺The two-photon fluorescent probe belongs to the field of organic fluorescent probes.

Background

Iron is a ubiquitous transition metal in the biosphere, essential for maintaining normal cell function due to its ability to participate in electron transfer reactions, however excess iron, especially redox-active Fe²⁺Toxic Reactive Oxygen Species (ROS) may also be generated, thereby damaging the body. Research has shown that oxidative stress caused by abnormal iron metabolism is closely related to some diseases such as Parkinson's Disease (PD). Based on the important position of iron in the diseases, the Fe in the cells can be rapidly and accurately detected²⁺The level is of great significance for researching pathogenesis of diseases such as PD and effectively monitoring occurrence and development of the diseases.

Therefore, real-time, non-invasive Fe was developed²⁺The specific detection tool is especially important for researching pathogenesis of related diseases such as PD. In order to solve the problem, research and development can be carried out to efficiently and accurately detect Fe²⁺The fluorescent probes of (a) will be of great value.

Commercial Fe²⁺Fluorescent probes have not emerged until recently, mainly two: FeRhoNox-1 and Ferrooorange. According to related reports, the probes can realize Fe²⁺But is expensive (1300 yuan/50 ug or 2480 yuan/24 ug), and due to the single photon excitation property, the penetration depth of the probes in the biological tissue is limited, which also affects the imaging effect of the probes in the organism. Meanwhile, data such as detection limits of these probes and disease application models are not clear.

Here, we proposeA two-photon fluorescent probe PyFe (excited by a single photon at 488nm or a two-photon at 880nm, and emitted at 630 nm) is disclosed, which can not only detect Fe²⁺Has extremely high selectivity, rapid response rate and extremely low theoretical detection limit (0.04nM) and can realize low concentration Fe²⁺The naked eye detection. The probe can realize imaging under single-photon excitation wavelength and two-photon excitation wavelength. Experiments prove that under the two-photon excitation, the probe can be imaged more clearly in a living body, the penetration depth in the brain tissue of a mouse can reach 391 mu m, and the probe can be applied to PD biological model imaging. In these respects, the performance of the PyFe probe is superior to that of the existing commercial probes.

Disclosure of Invention

The invention utilizes the optical and biological properties of the pyrimidine-based dye DEt and introduces Fe on the structure²⁺The N-O structure of the specific recognition group is further realized, thereby achieving the purpose of accurately detecting Fe in living cells²⁺Also can be used for detecting Fe in wild type and PD animal models²⁺。

In order to solve the technical problem of the invention, the technical scheme is as follows: fe²⁺The fluorescent probe is named as PyFe and has the following structural formula:

preferably, the optimal absorption wavelength of the probe is 360-370nm when the probe is in contact with Fe²⁺After the reaction, the absorption wavelength red shifts to 470-490nm, and the emission wavelength at this time is 620-640 nm.

Fe mentioned in the present invention²⁺Two-photon fluorescent probe and Fe²⁺The reaction mechanism of (a) is as follows:

in order to solve the technical problem of the invention, another technical scheme is provided: fe²⁺The preparation method of the two-photon fluorescent probe comprises the following steps: dissolving pyrimidine in appropriate amount of solventAfter the pyridine-based dye DEt, slowly adding m-chloroperoxybenzoic acid (m-CPBA) under an ice bath condition, stirring at room temperature for 0.5-3h, spinning off the solvent after the reaction is finished, and separating and purifying by a silica gel chromatography to obtain the target product PyFe fluorescent probe.

Preferably, the molar ratio of the pyrimidine-based dye DEt to the m-CPBA is 1: 1-1: 10.

preferably, the molar ratio of the pyrimidine-based dye DEt to the m-CPBA is 1: 2.2.

preferably, the preparation method of the pyrimidine-based dye DEt comprises the following steps: 4-fluorobenzaldehyde, potassium carbonate, diethylamine and Aliquat-336 are dissolved in DMF and stirred for 12h at 95 ℃. The reacted solution was dissolved in ethyl acetate and washed with water. Anhydrous sodium sulfate is used for removing water, and the DEt-a is obtained by silica gel chromatography purification.

Dissolving DEt-a in ethanol, ultrasonically dissolving the solid, adding concentrated hydrochloric acid while stirring, and refluxing at 95 ℃ for 24 hours. After the reaction, the solution was poured into ice water, and neutralized by adding sodium bicarbonate. By CH₂Cl₂And (4) drying the organic phase by using anhydrous sodium sulfate after twice extraction, removing the anhydrous sodium sulfate and the organic solvent, and obtaining the dye DEt by using a silica gel chromatography method.

Preferably, the invention relates to Fe²⁺The reaction formula for preparing the two-photon fluorescent probe is as follows:

the invention adopts another technical scheme that: one kind of Fe²⁺Application of two-photon fluorescent probe in precisely detecting Fe in organism in physiological system²⁺The preparation of the reagent (2).

Preferably, the reagent is used for accurately detecting Fe in organisms in a cell system through two-photon excitation regulation²⁺While avoiding interference from other ions and amino acids in the cell.

Preferably, the living cells and animal tissues are HepG2 cell line, LO2 cell line, zebrafish embryos, drosophila brains and C57BL/6 mice.

Has the advantages that:

the fluorescent probe has the advantages of near infrared emission and two-photon property, so that the fluorescent probe has the advantages of strong tissue penetration capability, weak light damage, weak influence of tissue autofluorescence and the like, and the fluorescence quantum yield and the two-photon absorption cross section are obviously improved after reaction. In vitro experimental studies show that the probe and Fe²⁺The reaction speed is high (<3min), low detection limit (0.04nM) and visible color change. For Fe under biological conditions (pH 7.4)²⁺Has high sensitivity and selectivity, and is not interfered by other ions and amino acids. Biological experimental studies show that the probe has low toxicity to nerve cells and has successfully achieved good imaging effect in various modes of organisms. The probe can detect exogenous and endogenous Fe in cells and zebra fish embryos²⁺The level of (c). The data comparison of single-photon and two-photon fluorescence imaging shows that the two-photon excitation characteristic can provide a clearer image and deeper tissue penetration depth. The probe is further applied to a PD animal model, and experiments prove that the probe can intuitively display endogenous Fe in PD drosophila brain tissue and PD mouse brain tissue²⁺And (4) horizontal.

FIG. 3: (a)10 μ M probe PyFe in DMSO, with 0-100nM Fe²⁺Absorption spectrum of the reaction; (b)10 μ M probe PyFe in DMSO, with 0-100nM Fe²⁺Emission spectrum of the reaction; (c) 10 μ M probes PyFe and 100nM Fe²⁺In DMSO, the reaction plateau can be reached within 3 min; (d) the 46 different ions and amino acids had little effect on the PyFe probe (1. Na)⁺,2.K⁺,3.Mg²⁺,4.Al³⁺,5. Fe³⁺,6.Mn²⁺,7.Cd²⁺,8.Zn²⁺,9.Ni²⁺,10.Cr³⁺,11.Al³⁺,12.Ca²⁺,13.Pd²⁺,14.Ag⁺, 15.Cu²⁺,16.Cu⁺,17.SO₄ ^2-,18.HCO₃ ^-,19.F^-,20.Br^-,21.I^-,22.ClO^-,23.CO₃ ^2-,24. SO₃ ^2-,25.HS^-,26.SCN^-,27.HPO₄ ^2-,28.H₂PO₄ ^-,29.Histidine,30.Proline,31. L-Alanine,32.L-Aspartic acid,33.L-Threonine,34.DL-methionine,35. L-tryptophan,36.L-serine,37.L-phenylalanine,38.L(+)-arginine,39.Tyrosine,40. DL-cys,41.L-isoleucine,42.Glycine,43.L-valine,44.L-ornithine,45.Glutamicacid, 46.DL-homocystine,47.Fe²⁺,48.Blank.)。

FIG. 4: when Fe is added to the probe²⁺Thereafter, the solution undergoes a macroscopic colour change

FIG. 5: (a) after 24 hours of action of PyFe with different concentrations, the survival rate of HepG-2 cells is more than 90% (MTT method); (b) after 24 hours of PyFe action at different concentrations, the survival rate of SH-SY5Y cells is more than 90% (MTT method).

FIG. 6: (a) PyFe can realize exogenous/endogenous Fe of HepG2 cells²⁺Horizontal fluorescence imaging, the fluorescence effect is good. Scale bar 10 μm; (b) relative fluorescence intensity (1-6). The statistical analysis used the t-test. P<0.001。

FIG. 7: (a) PyFe can realize exogenous/endogenous Fe in 5-day-old zebra fish²⁺Horizontal single-photon and two-photon fluorescence imaging is achieved, the fluorescence effect is good, and two-photon image signals are clearer. Scale bar 100 μm; (b) relative fluorescence intensity (2, 4,6, 8). The statistical analysis used the t-test. P<0.001。

FIG. 8: (a) PyFe can realize Fe in PD model drosophila melanogaster (Parkin null) and corresponding wild type drosophila melanogaster brain²⁺The horizontal two-photon fluorescence imaging has good fluorescence effect. Scale bar 50 μm; (b) relative fluorescence intensity (1-5). The statistical analysis used the t-test. P<0.001。

FIG. 9: (a) PyFe can realize Fe in PD and control group mouse brain²⁺Horizontal single-photon and two-photon fluorescence imaging has good fluorescence effect, and the penetration depth can reach 391 mu m. Scale bar 200 μm; (b) relative fluorescence intensity (3, 4). The statistical analysis used the t-test. P<0.01。

Drawings

The invention will be further explained with reference to the drawings.

FIG. 1: (a) nuclear magnetic hydrogen spectrum data of the probe PyFe; (b) nuclear magnetic carbon spectral data of PyFe probe

FIG. 2: nuclear magnetic mass spectrometry data for PyFe probe

FIG. 3: (a) probes and different concentrations of Fe²⁺Absorption spectrum after reaction; (b) probes and different concentrations of Fe²⁺Emission spectra after the reaction; (c) performing dynamic test on the probe; (d) selective testing of probes

FIG. 4: colour change of the probe under the naked eye

FIG. 5: (a) toxicity testing of the probes in HepG2 cells; (b) toxicity test of the Probe in SH-SY5Y cells

FIG. 6: (a) imaging of the probe in HepG2 cells; (b) corresponding relative fluorescence intensity

FIG. 7: (a) single-photon imaging and two-photon imaging of a probe in a live zebra fish embryo; (b) corresponding relative fluorescence intensity

FIG. 8: (a) two-photon imaging of probes in wild-type and parkinson's disease model drosophila brains; (b) corresponding relative fluorescence intensity

FIG. 9: (a) single and two-photon imaging of probes in control and parkinson model mouse brains; (b) corresponding relative fluorescence intensity

FIG. 10: reaction and application scheme of probe PyFe

Detailed Description

Example 1

Novel in vivo Fe detection method²⁺The preparation method of the two-photon fluorescent probe comprises the following steps:

1. preparation of pyrimidine-based dye DEt

4-fluorobenzaldehyde, potassium carbonate, diethylamine and Aliquat-336 are dissolved in DMF and stirred for 12h at 95 ℃. The reacted solution was dissolved in ethyl acetate and washed with water. Anhydrous sodium sulfate is used for removing water, and the DEt-a is obtained by silica gel chromatography purification.

2. Preparation of Probe PyFe

The pyrimidine-based dye DEt is applied to CHCl₃Dissolving, slowly adding m-CPBA under ice bath condition, and stirring for 3h at room temperature. And (3) after the reaction is finished, drying the solvent, and separating and purifying by a silica gel chromatography to obtain the target product PyFe fluorescent probe.

¹H NMR(400MHz,CD₃OD)δ7.88(d,J＝8.2Hz,6H),7.80(d,J＝7.7Hz,4H), 7.10(d,J＝15.1Hz,3H),3.85(s,4H),3.63(s,4H),1.06(d,J＝7.0Hz,12H).

¹³C NMR(101MHz,METHANOL-D4)δ159.01,149.37,138.00,136.23, 128.34,122.42,101.25,66.46,7.27.

Example 2

1. preparation of pyrimidine-based dye DEt

4-fluorobenzaldehyde, potassium carbonate, diethylamine and Aliquat-336 are dissolved in DMF and stirred for 14h at 95 ℃. The reacted solution was dissolved in ethyl acetate and washed with water. Anhydrous sodium sulfate is used for removing water, and the DEt-a is obtained by silica gel chromatography purification.

Dissolving DEt-a in ethanol, dissolving the solid by ultrasonic, adding concentrated hydrochloric acid while stirring, and refluxing at 95 ℃ overnight. After the reaction, the solution was poured into ice water, and neutralized by adding sodium bicarbonate. By CH₂Cl₂And (4) drying the organic phase by using anhydrous sodium sulfate after twice extraction, removing the anhydrous sodium sulfate and the organic solvent, and obtaining the dye DEt by using a silica gel chromatography method.

2. Preparation of Probe PyFe

The pyrimidine-based dye DEt is applied to CHCl₂Dissolving, slowly adding m-CPBA under ice bath condition, and stirring for 1h at room temperature. And (3) after the reaction is finished, drying the solvent, and separating and purifying by a silica gel chromatography to obtain the target product PyFe fluorescent probe.

Example 3

1. preparation of pyrimidine-based dye DEt

4-fluorobenzaldehyde, potassium carbonate, diethylamine and Aliquat-336 are dissolved in DMF and stirred for 20h at 95 ℃. The reacted solution was dissolved in ethyl acetate and washed with water. Anhydrous sodium sulfate is used for removing water, and the DEt-a is obtained by silica gel chromatography purification.

2. Preparation of Probe PyFe

The pyrimidine-based dye DEt is applied to CHCl₃Dissolving, slowly adding m-CPBA under ice bath condition, and stirring at room temperature for 0.5 h. And (3) after the reaction is finished, drying the solvent, and separating and purifying by a silica gel chromatography to obtain the target product PyFe fluorescent probe.

Example 4

Novel in vivo Fe detection method²⁺The two-photon fluorescence probe pair Fe²⁺In vitro response test of (1):

1. PyFe for different Fe²⁺Fluorescence response at concentration (fig. 3a, b):

first, 10. mu.M PyFe probe was added to DMSO, and then 0-100nM Fe was added²⁺Testing by fluorescence spectrophotometry, and drawing PyFe probe for different concentrations of Fe²⁺The fluorescence spectrum of (2) was observed, and PyFe probe was observed for Fe of 0 to 100nM²⁺With linear increaseStrong fluorescence response.

2. PyFe to Fe²⁺Reaction kinetics test (FIG. 3c)

First, 10. mu.M PyFe probe was added to DMSO, and then 100nM Fe was added²⁺Mixing uniformly, taking out 80 μ L to 384-well plate rapidly, detecting with enzyme-labeling instrument for a long time, and plotting PyFe to Fe²⁺The reaction kinetics curve of (A) shows that PyFe probe is specific to Fe²⁺Has extremely fast response speed.

3. Fluorescent response of PyFe to common ionic amino acids (fig. 3 d):

first, 10. mu.M PyFe probe was added to DMSO, and 47 ionic amino acids (100. mu.M) were added, respectively, and then the emission value of the reaction solution was measured at 630 nm. And drawing the fluorescence spectrogram of PyFe probe for different analytes to observe the PyFe probe for Fe²⁺It is selective, and ions/amino acids common in other organisms do not respond to the probe.

4. PyFe to Fe²⁺Detection limit of (2)

By detecting PyFe with Fe concentration gradient from 0-100nM²⁺The fluorescence intensity at 630nm was linearly fitted to the change in fluorescence after the response to obtain a calibration curve. PyFe to Fe is calculated according to the detection limit calculation formula (3 sigma/S)²⁺The detection limit of (a) is 0.04 nM.

Example 5

Novel in vivo Fe detection method²⁺The two-photon fluorescence probe pair Fe²⁺In vivo response detection of (3):

1. toxicity test of PyFe in different cells (fig. 5):

HepG2/SH-SY5Y cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100.0mg/mL streptomycin and 100 IU/mL penicillin. Cells were maintained in humidified air at 37 ℃ in 5% carbon dioxide.

The cytotoxicity of PyFe was determined using 3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl-2-H-tetrazolium ammonium bromide (MTT) colorimetric cell proliferation kit (Roche). HepG2/SH-SY5Y cells were harvested after 70-80% growth in 96-well plates, the medium was aspirated and then replaced with medium containing PyFe at various concentrations.

After 24h of incubation, 10. mu.L of MTT solution (5mg/mL) was added to the cells. After further incubation at 37 ℃ for 4h in the dark, the resulting crystals were dissolved by replacing the solution with 100. mu.L of dimethyl sulfoxide. After 10 minutes, the absorbance was measured at 560nm with a microplate reader, and the cell activity was plotted.

After 24h incubation with 30/50 μ M PyFe, HepG2 and SH-SY5Y cells all maintained more than 90% viability, indicating that PyFe has lower cytotoxicity at the working concentration.

2. Imaging assay of PyFe in HepG2 cells (fig. 6):

HepG2 cells at 5X 10⁵The density of individual cells was seeded in 35mm confocal laser imaging dishes and grown for 24 hours. PyFe probe for cell culture was incubated at 10. mu.M concentration and 5% CO at 37 ℃₂The atmosphere was maintained for 3h, then the cells were washed with PBS (3X 2ml per dish).

For exogenous experiments, cells were incubated with 50. mu.M Fe at 37 deg.C²⁺After incubation for 0.5h, followed by 3 washes with PBS, the cells were incubated for a further 2.5h with 10 μ M probe PyFe. (FIG. 6-a4)

To confirm that the fluorescence enhancement was due to intracellular Fe²⁺We applied Fe²⁺The chelating agent, 22' -bipyridine (Bpy), served as a control. (FIG. 6-a6)

All cells were washed 3 times with PBS before imaging. Finally, cells were imaged with a confocal laser scanning microscope (Zeiss LSM 880) and single photon laser, excitation wavelength 488nm, and recorded emission wavelength 570-700 nm.

The fluorescence intensity of the Image of the cells was quantified using Image J, and the mean fluorescence intensity values of each Image were taken and a t-test was performed between the values. (FIG. 6-b)

The experimental result shows that PyFe has good cell permeability, and the accurate detection of Fe in cells is realized²⁺Without interference from other materials in the cytoplasm.

3. Imaging test of PyFe in zebrafish (fig. 7):

the collected zebra fish eggs were cultured in E3 culture medium and divided into 4 groups after 5 days of age.

For exogenous experiments, the normal culture solution for culturing zebra fish was replaced with a solution containing 50. mu.M Fe²⁺The E3 culture solution was cultured at 25 ℃ for 1 hour, and then the culture solution containing 20. mu.M of the probe PyFe was used instead, and the culture was continued at 25 ℃ for 1 hour. (FIGS. 7-a5/6)

All zebrafish were washed 3 times with PBS prior to imaging. And finally, imaging the zebra fish by using a confocal laser scanning microscope (Zeiss LSM 880) and single-photon and two-photon lasers, wherein the single-photon excitation wavelength is 488nm, the two-photon excitation wavelength is 880nm, and the recorded emission wavelength is 570-700 nm.

The fluorescence intensity of the Image of the cells was quantified using Image J, and the mean fluorescence intensity values of each Image were taken and a t-test was performed between the values. (FIG. 7-b)

The experimental result shows that PyFe can realize the detection of Fe in vivo of living animals²⁺The two-photon imaging can present clearer image details and has good imaging effect.

Example 6

1. Imaging test of PyFe in wild type and PD model drosophila brain tissue (figure 8):

wild-type and PD-type drosophila were from the neurodegeneration research laboratory of the national neuroscience institute of singapore.

All flies were grown on corn meal-molasses medium and maintained at 25 ℃, PD and wild type flies were identified as experimental (fig. 8-a2, fig. 8-a5) and control (fig. 8-a1, fig. 8-a4), respectively, and Bpy-added flies were used as the control (fig. 8-a 3). After dissection, the drosophila brain tissue of the experimental group was placed in PBS (pH 7.4) buffer containing 20 μ M PyFe. The drosophila brain tissue of the suppression group was immersed in PBS buffer containing Bpy (1mM), incubated at 37 ℃ for 1 hour, then immersed in PBS buffer containing 20 μ M probe PyFe, and incubated at 37 ℃ for an additional 2 hours. Subsequently, each group of brain tissue was washed 3 times with PBS.

Finally, a confocal laser scanning microscope (Zeiss LSM 880) and a two-photon laser are used for imaging the drosophila brain tissue, wherein the two-photon excitation wavelength is 880nm, and the recorded emission wavelength is 570-700 nm.

Fluorescence intensity quantification of Drosophila brain imaging images was performed using Image J, and the mean fluorescence intensity values for each Image were taken and a t-test was performed between the values. (FIG. 8-b)

Experimental results show that PyFe has good imaging performance in brain tissues of PD insect models.

2. Imaging test of PyFe in PD model mouse brain tissue (fig. 9):

all mice were randomly divided into two groups (control group and experimental group), and mice in the experimental group were intraperitoneally injected with MPTP (25mg/kg) dissolved in physiological saline every day for 10 consecutive days; control mice were injected daily with an equal amount of sterile saline.

And after the PD model is constructed, performing behavioral verification.

The brains of PD mice and control mice were then carefully isolated. Brain sections were obtained with a vibrating microtome. Brain pieces (thickness 500 μ M) were incubated with PyFe probe (50 μ M), 1% DMSO, and 0.1% Triton in PBS (pH 7.4) for 2 hours.

Subsequently, each group of sections was washed three times with PBS. And finally, imaging the brain tissue of the drosophila by using a confocal laser scanning microscope (Zeiss LSM 880) and single-photon and two-photon lasers, wherein the single-photon excitation wavelength is 488nm, the two-photon excitation wavelength is 880nm, and the recorded emission wavelength is 570-700 nm.

Fluorescence intensity quantification of Drosophila brain imaging images was performed using Image J, and the mean fluorescence intensity values for each Image were taken and a t-test was performed between the values. (FIG. 9-b)

Experiments show that the probe PyFe can detect the iron enrichment condition in the brain of the PD model mouse. In a tissue sample with a certain thickness, the penetrating depth of 391 mu m can be achieved, and the presented image is clear and is suitable for animal tissue imaging.

The invention is not limited to the specific technical solutions described in the above embodiments, and all technical solutions formed by equivalent substitutions are within the scope of the invention as claimed.

Claims

1. Fe²⁺The two-photon fluorescent probe is characterized in that: the fluorescent probe is PyFe probe, PThe structure of the yFe fluorescent probe is shown as the formula (I):

2. fe according to claim 1²⁺The two-photon fluorescent probe is characterized in that: the optimal absorption wavelength of the probe is 360-370nm when the probe and Fe are mixed²⁺After the reaction, the absorption wavelength red shifts to 470-490nm, and the emission wavelength at this time is 620-640 nm.

3. Fe according to claim 1²⁺The preparation method of the two-photon fluorescent probe is characterized by comprising the following steps: the method comprises the following steps: dissolving the pyrimidine-based dye DEt in a proper amount of solvent, slowly adding m-chloroperoxybenzoic acid (m-CPBA) under an ice bath condition, stirring at room temperature for 0.5-3h, drying the solvent after the reaction is finished, and separating and purifying by a silica gel chromatography to obtain the target product PyFe fluorescent probe.

4. Fe according to claim 3²⁺The preparation method of the two-photon fluorescent probe is characterized by comprising the following steps: the molar ratio of the pyrimidine-based dye DEt to the m-CPBA is 1: 1-1: 10.

5. fe according to claim 3²⁺The preparation method of the two-photon fluorescent probe is characterized by comprising the following steps: the preparation method of the pyrimidine-based dye DEt comprises the following steps: dissolving 4-fluorobenzaldehyde, potassium carbonate, diethylamine and methyl trioctyl ammonium chloride (Aliquat-336) in DMF, and stirring at 95 ℃ for 12 h; dissolving the solution after reaction by using ethyl acetate, and washing by water; anhydrous sodium sulfate is used for removing water, and the DEt-a is obtained by silica gel chromatography purification. Dissolving DEt-a and 2-hydroxy-4, 6-dimethylpyrimidine in ethanol, ultrasonically dissolving solids, stirring, adding concentrated hydrochloric acid, and refluxing at 95 ℃ for 24 hours; after the reaction is finished, pouring the solution into ice water, and adding sodium bicarbonate for neutralization; by CH₂Cl₂After two extractions, the organic phase is dried over anhydrous sodium sulfate, and the anhydrous sodium sulfate and the organic solvent are removedAnd obtaining the dye DEt by a silica gel chromatography method.

6. Fe according to claim 3²⁺The preparation method of the two-photon fluorescent probe is characterized by comprising the following steps: the preparation reaction scheme is as follows:

7. an Fe according to claim 1²⁺The application of the two-photon fluorescent probe is characterized in that: for preparing Fe for accurately detecting Fe in organisms in physiological systems²⁺The detection reagent of (1).

8. Fe according to claim 7²⁺The application of the two-photon fluorescent probe is characterized in that: the detection reagent can accurately detect Fe in a living body in living cells or animal tissues through single photon or two-photon excitation regulation²⁺While avoiding interference from other ions and amino acids in the cell.

9. An Fe according to claim 8²⁺The application of the two-photon fluorescent probe is characterized in that: the living cells and animal tissues are HepG2 cell lines, LO2 cell lines, zebrafish embryos, drosophila cerebrums or C57BL/6 type mice.