阅读说明：本技术 一种Zn2+离子激活型荧光探针 (Zn2+Ion-activated fluorescent probe ) 是由 赵梦 李飞 朱夏夏 黄山 于 2021-01-19 设计创作，主要内容包括：本发明提供一种Zn～(2+)离子激活型荧光探针及其应用,涉及激活型荧光探针设计合成与应用技术领域。所述激活型小分子荧光探针,能够专一性检测体内外Zn～(2+)离子水平,实现激活型荧光成像,且不受其他金属离子的干扰。同时,本发明中目标探针与Zn～(2+)离子作用后,荧光强度迅速的实现“OFF”到“ON”的转变,可以应用于体内外Zn～(2+)离子的快速荧光检测。本发明采用一步合成目标探针的制备方法,可操作性强,简单高效,为Zn～(2+)离子异常相关疾病尤其是AD的诊断和治疗提供潜在的应用价值。(The invention provides Zn 2+ An ion-activated fluorescent probe and application thereof relate to the technical field of activated fluorescent probe design synthesis and application. The activated small molecular fluorescent probe can specifically detect Zn inside and outside a body 2+ And (4) ion level, so that activated fluorescence imaging is realized, and interference of other metal ions is avoided. Meanwhile, the target probe of the present invention is Zn 2+ After the ion action, the fluorescence intensity can rapidly realize the conversion from OFF to ON, and can be applied to in vivo and in vitro Zn 2+ Rapid fluorescence detection of ions. The preparation method for synthesizing the target probe by one step has strong operability, is simple and efficient and is Zn 2+ The diagnosis and treatment of diseases associated with ion abnormalities, particularly AD, provide potential application value.)

1. Zn²⁺An ion-activated fluorescent probe, characterized in that Zn is contained in the probe²⁺The chemical structure of the ion-activated fluorescent probe is shown as follows:

2. zn according to claim 1²⁺An ion-activated fluorescent probe characterized in that: said Zn²⁺The preparation method of the ion-activated fluorescent probe comprises the following steps: and (3) carrying out nucleophilic addition-elimination reaction on the 4-diethylamino salicylaldehyde and p-methylaniline to obtain the target probe TN.

3. A Zn according to claim 2²⁺An ion-activated fluorescent probe characterized in that: the nucleophilic addition-elimination reaction is carried out in absolute ethyl alcohol; and the mol ratio of the 4-diethylamino salicylaldehyde to the methylaniline is 1: 1.

4. A Zn according to claim 2²⁺An ion-activated fluorescent probe characterized in that: the nucleophilic addition-elimination reaction adopts glacial acetic acid as a reaction catalyst, the reaction is a reflux reaction for 5 hours, and recrystallization is adopted to purify the target probe.

5. Zn as claimed in claim 1²⁺Use of an ion-activated fluorescent probe, said Zn²⁺The application of the ion-activated fluorescent probe mainly comprises the following steps: in the detection of Zn²⁺Application in ions; in the preparation of Zn²⁺The application in ion activated reagent kit; fluorescence in cellsApplications in light imaging; the application in preparing cell fluorescence imaging reagent.

6. A Zn according to claim 5²⁺Use of an ion-activated fluorescent probe, wherein Zn is present²⁺The application mode of the ion-activated fluorescent probe in cell fluorescence imaging is as follows: zn is added²⁺Adding the ion-activated fluorescent probe solution into normal cells or Zn²⁺And (3) in the cells subjected to ion pretreatment, after culturing and hatching, absorbing the culture solution, then adding a buffer solution, and performing fluorescence detection to finish cell imaging.

7. A Zn according to claim 6²⁺Use of an ion-activated fluorescent probe, wherein Zn is present²⁺The solvent in the ion-activated fluorescent probe solution is a 1% DMSO aqueous solution.

8. A Zn according to claim 6²⁺The application of the ion-activated fluorescent probe is characterized in that the cells are selected from human liver cancer HepG2 cells.

Technical Field

The invention relates to the technical field of design synthesis and application of an activated fluorescent probe, in particular to Zn²⁺An ion-activated fluorescent probe and applications thereof.

Background

With the increase of global aging, the ratio of the aged populationFor example, as the number of years increases, the problems caused by Alzheimer's Disease (AD for short) become more serious. Extracellular β -amyloid plaques (β -amyloid plaques) formed by β -amyloid (β -amyloid, a β protein) in the brain are one of the most important pathological features of alzheimer's disease. Neurotoxicity of A beta plaque in brain and abnormal concentration of Zn in A beta plaque²⁺Ions being closely related, i.e. Abeta plaques with Zn²⁺Abnormal binding of ions and the like will lead to the formation of a β plaques and produce irreversible neurotoxicity. Abeta protein and Zn²⁺Ionic interactions are an important factor in the development of AD.

Zn²⁺The ion is taken as the second most abundant transition metal ion in human bodies and other mammals, and plays an important role in a plurality of biological processes such as apoptosis regulation, signal transmission, enzyme function, gene expression and the like. Currently, Zn is being detected in numerous ways²⁺In the ion analysis method, small molecular fluorescent probes attract extensive attention due to high sensitivity and high specificity, but the probes still have high detection limit and are difficult to detect low-dose Zn²⁺And meanwhile, the molecular weight of the probe is large, the physiological toxicity is high, and subsequent biological application cannot be carried out. Therefore, a novel small molecular fluorescent probe is designed and synthesized to detect Zn at physiological level²⁺The ion concentration is used for exploring the action mechanism of the AD in the generation process, and the method has important scientific research and clinical values.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides Zn²⁺Ion-activated fluorescent probes and their use, Zn²⁺The ions can effectively activate the fluorescent signal of the probe, realize the conversion of the fluorescence from OFF to ON, and utilize the characteristics of the activated imaging to carry out the intracellular Zn²⁺And ion imaging lays a foundation for later exploration of interaction of Zn2+ ions and A beta protein.

In order to achieve the above purpose, the technical scheme of the invention is realized by the following technical scheme:

zn²⁺Ion-activated fluorescent probe, said Zn²⁺The chemical structure of the ion-activated fluorescent probe is shown as follows:

preferably, the Zn is²⁺The preparation method of the ion-activated fluorescent probe comprises the following steps: and (3) carrying out nucleophilic addition-elimination reaction on the 4-diethylamino salicylaldehyde and p-methylaniline to obtain the target probe TN.

Preferably, the nucleophilic addition-elimination reaction is carried out in absolute ethanol; and the mol ratio of the 4-diethylamino salicylaldehyde to the methylaniline is 1: 1.

Preferably, glacial acetic acid is used as a reaction catalyst in the nucleophilic addition-elimination reaction, the reaction is a reflux reaction for 5 hours, and recrystallization is adopted to purify the target probe.

Said Zn²⁺The application of the ion-activated fluorescent probe mainly comprises the following steps: in the detection of Zn²⁺Application in ions; in the preparation of Zn²⁺The application in ion activated reagent kit; the application in cell fluorescence imaging; the application in preparing cell fluorescence imaging reagent.

Preferably, the Zn is²⁺The application mode of the ion-activated fluorescent probe in cell fluorescence imaging is as follows: zn is added²⁺Adding the ion-activated fluorescent probe solution into normal cells or Zn²⁺And (3) in the cells subjected to ion pretreatment, after culturing and hatching, absorbing the culture solution, then adding a buffer solution, and performing fluorescence detection to finish cell imaging.

Preferably, the Zn is²⁺The solvent in the ion-activated fluorescent probe solution is a 1% DMSO aqueous solution.

Preferably, the cells are selected from human liver cancer HepG2 cells.

The invention provides Zn²⁺Compared with the prior art, the ion-activated fluorescent probe and the application thereof have the advantages that:

(1) the invention designs and synthesizes a novel activated small-molecule fluorescent probe, and a one-step synthesis method is adopted for synthesis, so that the method is simple and efficient;

(2) target probe Zn in the invention²⁺After ion recognition, the fluorescence intensity realizes the conversion from OFF to ON, and the activated fluorescence imaging is realized.

(3) Target probe in the invention is related to Zn in cell²⁺After ion co-incubation, intracellular Zn is realized²⁺Ion activated fluorescence imaging.

Drawings

FIG. 1 shows Zn of the present invention²⁺A schematic diagram of the synthesis of an ion-activated fluorescent probe;

FIG. 2(a) is a diagram showing UV-VIS absorption spectra of a target probe TN in different solvents; (b) fluorescence emission spectrograms of the target probe TN in different solvents;

FIG. 3(a) shows the UV-VIS absorption spectrum of the target probe TN in the presence of different metal ions; (b) fluorescence emission spectrum of target probe TN in the presence of different metal ions;

FIG. 4(a) is a graph showing the result of cytotoxicity test of TN as a target probe; (b) a light stability test result graph of the target probe TN is shown;

FIG. 5 is a diagram of confocal fluorescence imaging of living cells of the target probe TN.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described below in conjunction with the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1:

Zn²⁺synthesizing and characterizing the ion-activated fluorescent probe:

(1) a50 mL round-bottom flask was charged with p-methylaniline 0.54g (107.15g/mol,5.0mmol), 4-diethylamino-salicylaldehyde 0.97g (193.24g/mol,5.0mmol) and 20mL of anhydrous ethanol as solvent, followed by dropwise addition of 1mL of glacial acetic acid as catalyst, and the mixture was magnetically stirred and refluxed for 5 h. After the reaction is finished, cooling to room temperature, precipitating a yellow solid, performing suction filtration to obtain a solid, recrystallizing with 20mL of ethanol to obtain the target probe TN, wherein the synthetic schematic diagram is shown in FIG. 1.

Example 2:

Zn²⁺establishment of solvation ultraviolet visible absorption spectrum and fluorescence emission spectrum of the ion-activated fluorescent probe:

(1) the target probe TN prepared in example 1 was dissolved in a DMSO solution and prepared at a concentration of 1X 10^-3mol/L mother liquor;

(2) adding 50 mu L of mother liquor into 4500 mu L of chromatographic pure solvent respectively, wherein the sequence of the polarity of the solvent from low to high is as follows: benzene, dichloromethane, tetrahydrofuran, ethyl acetate, ethanol, acetonitrile, N-dimethylformamide;

(3) and (3) taking a proper amount of the sample, and carrying out ultraviolet visible absorption spectrum and fluorescence emission spectrum tests on a quartz cuvette of 1 cm.

The ultraviolet-visible absorption spectrum was measured using a SPECORD S600 spectrophotometer, the fluorescence emission spectrum was measured using a Hitachi F-7000 fluorescence spectrophotometer, and the ultraviolet-visible spectrum change was measured using an ultraviolet-visible spectrophotometer, and the results are shown in FIG. 2 (a).

The result shows that the maximum absorption wavelength of the target probe TN in solvents with different polarities is 350-400 nm, and the optimal absorption wavelength is slightly red-shifted along with the increase of the polarity of the solvents, so that the obvious solvation effect is presented.

Since the polarity of the molecule in the excited state is greater than that in the ground state, the polar solvent lowers the energy of both the excited state and the ground state, but the excited state is lowered more, and thus the electron absorption energy of the molecule is reduced as compared with that of the nonpolar solvent, so that the absorption band is red-shifted. FIG. 2(b) is a fluorescence emission spectrum measured under the same conditions. The result shows that the fluorescence emission intensity of the target probe TN is weak, the optimal emission wavelength is between 400nm and 550nm, and the fluorescence intensity and the emission wavelength show irregular changes under the solvent polarity image.

Example 3:

Zn²⁺ion activated typeEstablishing an ultraviolet visible absorption spectrum and a fluorescence emission spectrum of the fluorescent probe in the presence of different metal ions:

(1) the target probe TN prepared in example 1 was dissolved in a DMSO solution to prepare a mother solution having a concentration of 1X 10-3 mol/L.

(2) Adding 50 μ L of the mother liquor into 4925 μ L of the aqueous solution, and adding 25 μ L of the mother liquor, 1 × 10^-2Solutions of different metal ions in mol/L in nitrates (Blank, Fe)³⁺、Ni²⁺、Al³⁺、Co²⁺、Cu²⁺、Ca²⁺、Bi³⁺、Cr³⁺、Li⁺、Pb²⁺、Na⁺、Ba²⁺、Zn²⁺、Cd²⁺、K⁺、Hg²⁺、Mn²⁺、Fe²⁺And Mn²⁺Ions) so that the final concentration of metal ions is 5 × 10^-5mol/L。

(3) And (3) taking a proper amount of the sample, and carrying out ultraviolet visible absorption spectrum and fluorescence emission spectrum tests on a quartz cuvette of 1 cm.

The UV-visible absorption spectrum was measured using a SPECORD S600 spectrophotometer, and the fluorescence emission spectrum was measured using a Hitachi F-7000 fluorescence spectrophotometer.

As shown in FIG. 3(a), since the O atom of the phenolic hydroxyl group and the N atom of the C ═ N bond on the TN molecule of the target probe have a certain coordination function, and the zinc ion as a transition metal element has a vacant orbital capable of coordination, the energy level is changed during the coordination process, and the absorption peak is changed, the Zn is added compared with the blank control without any ion²⁺After the ion, a new obvious shoulder peak appears at 350nm of the target probe TN, and the original main peak position generates red shift.

FIG. 3(b) is a fluorescence emission spectrum measured under the same conditions, and the results show that TN as a target probe is Zn only²⁺The fluorescence is obviously enhanced in the presence of the ions, and other metal ions do not have obvious change on the change of the fluorescence intensity, which indicates that the probe TN is indeed Zn²⁺An ion activated fluorescent probe.

Example 4:

Zn²⁺cytotoxicity test and photostability test of ion-activated fluorescent probe:

1. cytotoxicity experiments:

(1) HepG2 cells in log phase growth phase were seeded in flat-bottomed 96-well plates in a medium containing 5% CO₂Incubating for 24 hours in a constant temperature incubator at 37 ℃;

(2) diluting the probe TN to the required concentration (0, 10, 20, 40, 60 and 80 mu M) by using fresh DMEM medium, absorbing the original medium in a 96-well plate, respectively adding 100 mu L of probe solution diluted by the medium with different concentrations, and continuously incubating for 24 hours at 37 ℃;

(3) after the probe incubation was complete, the original medium was aspirated, each well was washed twice with 100. mu.L of cold PBS, and 100. mu.L of MTT solution diluted in medium (0.5mg/mL) was added in 5% CO₂Incubating for 4 hours in a constant temperature incubator at 37 ℃;

(4) after incubation is finished, carefully absorbing the solution in a 96-well plate, adding 100 mu L of dimethyl sulfoxide solution, shaking at normal temperature for 5min, and then measuring by using an enzyme-labeling instrument;

(5) and (3) taking the absorbance of the cell population which is not processed by the probe under the same experimental condition as a reference, calculating the cell survival rate by using a formula, and repeatedly measuring the experiment for three times.

From fig. 4(a), it can be seen that the survival rate of cells reaches above 85% in the presence of lower concentration of the compound, and the lower toxicity can be used for the subsequent intracellular Zn2+ ion-activated confocal fluorescence imaging.

2. Photostability experiment: fluorescence intensity of 5 μ M probe TN in an aqueous solution was measured using fluorescence spectroscopy, laser irradiation was performed for 60 seconds per minute, 10 times of continuous irradiation, and 10 changes in fluorescence intensity were recorded.

FIG. 4(b) shows that the probe has good light stability in 10min, and the fluorescence intensity of the probe still remains over 90% of the initial intensity within 10 min.

Example 5:

Zn²⁺confocal fluorescence imaging of ion-activated fluorescent probes:

(1) the target probe TN obtained in example 1 above was dissolved in water containing 1% DMSO, added to HepG2 cell culture dishes (5. mu.M) having a cell enrichment degree of 60%, placed in a constant temperature incubator to incubate for 30min, followed by aspiration of the culture solution and washing twice with PBS buffer (2X 1mL), and finally 1mL of PBS buffer was injected into each well.

(2)Zn²⁺In ion activation experiments, Zn²⁺After the ions had been co-cultured with HepG2 cells for 30min (20. mu.M) in advance, the culture solution was washed away, the medium was changed to TN (5. mu.M) and co-cultured for 30min, the culture solution was washed away, and washed twice with PBS buffer (2X 1mL), and finally the dishes were filled with 1mL of PBS buffer and confocal imaging was performed.

As shown in fig. 5, and without Zn addition²⁺Compared with the control group of ions, the experimental group has remarkably enhanced intracellular fluorescence signals, and the signal region is concentrated in cytoplasm, which indicates that Zn is contained in cells²⁺The ions can effectively activate the fluorescent signal of the probe TN, and the intracellular activation type fluorescence imaging is realized.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.