阅读说明：本技术 一种锌铝离子识别荧光探针及其制备方法和应用 (Zinc-aluminum ion recognition fluorescent probe and preparation method and application thereof ) 是由 李培勇 芦鑫淼 秦敬灿 朱新远 于 2021-06-16 设计创作，主要内容包括：本发明提供一种锌铝离子识别荧光探针及其制备方法和应用,荧光探针分子内含有席夫碱结构及三个苄基集团。本发明提供的锌铝离子荧光探针具有双机制、双响应、高灵敏度性和高特异性、不受其他金属离子影响、易被细胞摄取、细胞毒性低等特点。将该探针应用于前列腺癌患者的外周血,可以对血清中的锌含量进行定量检测,为疑似前列腺癌患者的诊断提供宝贵信息。将其与前列腺癌血清PSA检测结合,可以有效提升前列腺癌的诊断准确性,避免血清PSA处于灰度区间内的患者的穿刺诊断需要,进而避免过度诊断和过度治疗的发生。(The invention provides a zinc-aluminum ion recognition fluorescent probe and a preparation method and application thereof. The zinc-aluminum ion fluorescent probe provided by the invention has the characteristics of double mechanisms, double response, high sensitivity, high specificity, no influence of other metal ions, easy cell uptake, low cytotoxicity and the like. The probe is applied to peripheral blood of a prostate cancer patient, can quantitatively detect the zinc content in serum, and provides valuable information for diagnosis of a suspected prostate cancer patient. The kit is combined with prostate cancer serum PSA detection, so that the diagnosis accuracy of prostate cancer can be effectively improved, the puncture diagnosis requirement of a patient with serum PSA in a gray scale interval is avoided, and the occurrence of over-diagnosis and over-treatment is further avoided.)

1. A zinc-aluminum ion recognition fluorescent probe is characterized in that the structural formula is as follows:

2. the preparation method of the zinc-aluminum ion recognition fluorescent probe of claim 1, which is characterized by comprising the following steps: the probe is prepared by performing substitution reaction on ethyl 3,4, 5-trihydroxybenzoate and benzyl chloride under the condition that potassium carbonate is used as an acid-binding agent to obtain methyl 3,4, 5-tribenzyloxy benzoate, then performing hydrazinolysis on the methyl 3,4, 5-tribenzyloxy benzoate to obtain 3,4, 5-tribenzyloxy benzoyl hydrazine, and performing condensation reaction on the 3,4, 5-tribenzyloxy benzoyl hydrazine and salicylaldehyde.

3. The use of the zinc-aluminum ion recognition fluorescent probe of claim 1 in detecting serum zinc-aluminum content.

Technical Field

The invention relates to the technical field of fluorescent probes, in particular to a zinc-aluminum ion recognition fluorescent probe and a preparation method and application thereof.

Background

Prostate cancer is the most prevalent cancer in men, and there has been a clear trend toward younger incidence in recent years. In a normal prostate, the glandular cells are structurally intact and the glands function normally, and the PSA level in serum is generally below 1 ng/mL. However, when prostate cancer develops, the morphology and structure of prostate cells and tissues are destroyed, resulting in leakage of cell surface antigens into the serum and elevated serum PSA levels. Thus prostate cancer patients generally have higher serum PSA levels than normal patients. Although serum PSA levels are considered as important diagnostic biomarkers for prostate cancer, the clinical use of serum PSA assays is limited due to their lack of certainty and specificity in diagnosing prostate cancer, as they are also accompanied by elevated serum PSA levels during benign prostate conditions (e.g., prostatic hyperplasia, local inflammation, infection, etc.).

After the prostate gland becomes cancerous, the zinc content in the gland and in the serum is significantly reduced, whereas in non-cancerous lesions no significant change in the serum zinc content is observed. The detection of the zinc level in the serum can be used as a complementation and reference for the evaluation of the PSA content in the serum, and can be used for realizing the accurate diagnosis of the prostate cancer. There are currently a number of ways to detect free zinc, with inductively coupled plasma mass spectrometry (ICP-MS) being the most common way. However, the detection mode is limited by expensive instruments and detection price, and has difficulty in popularization and application in clinical processes. Fluorescent probes are attracting more and more attention in medical diagnosis and detection processes due to their advantages, such as low cost, simple operation, and visual detection.

There are also a number of zinc-responsive fluorescent probes, most of which have a 2-picolinamino group that is specifically coordinated to zinc recognition. The group is installed in a fluorescent molecule, the fluorescence of the fluorescent molecule is quenched by the photoinduced electron transfer effect (PET) between the group and the fluorescent molecule, and when the recognition group is specifically coordinated with free zinc ions, the PET effect is blocked, and the fluorescence is recovered. The zinc ions can be quantitatively detected by recovering the fluorescence intensity of the fluorescence. However, most of such probes have an aggregate fluorescence quenching (ACQ) effect, i.e., fluorescence is quenched in a solid state and an aggregate state, which affects the accuracy of the detection result. Aggregation-induced emission (AIE) fluorescence is a molecule which does not generate fluorescence or generates weak fluorescence in a disperse system but can generate obvious fluorescence in an aggregation state, and the development of the zinc-responsive AIE fluorescent probe can effectively avoid fluorescence quenching caused by aggregation of the fluorescent probe due to concentration, intermolecular interaction and the like in the detection process and improve the detection accuracy.

Disclosure of Invention

The first purpose of the invention is to provide a dual-mechanism and dual-response zinc-aluminum ion recognition fluorescent probe.

The second purpose of the invention is to provide a preparation method of the zinc-aluminum ion recognition fluorescent probe with double mechanisms and double responses.

The third purpose of the invention is to provide the application of the zinc-aluminum ion recognition fluorescent probe with double mechanisms and double responses.

In order to achieve the first object, the present invention provides a zinc-aluminum ion recognition fluorescent probe (3,4, 5-Tris-phenyloxy-benzoic acid (2-hydroxy-phenylidene) -hydrazide, abbreviated as HL), which has a structural formula:

in order to achieve the second object, the invention provides a preparation method of a zinc-aluminum ion recognition fluorescent probe, which comprises the following steps: the probe is prepared by performing substitution reaction on ethyl 3,4, 5-trihydroxybenzoate and benzyl chloride under the condition that potassium carbonate is used as an acid-binding agent to obtain methyl 3,4, 5-tribenzyloxy benzoate, then performing hydrazinolysis on the methyl 3,4, 5-tribenzyloxy benzoate to obtain 3,4, 5-tribenzyloxy benzoyl hydrazine, and performing condensation reaction on the 3,4, 5-tribenzyloxy benzoyl hydrazine and salicylaldehyde.

In order to achieve the third purpose, the invention provides an application of the zinc-aluminum ion recognition fluorescent probe in detecting the content of zinc and aluminum in serum.

The fluorescent probe HL molecule of the invention contains a Schiff base structure and three benzyl groups. In an ethanol solvent, the Schiff base unit can quench the fluorescence of the fluorescent probe through the action of Photoinduced Electron Transfer (PET), and when the Schiff base unit and aluminum ions are combined and coordinated in a 1:1 mode, the PET effect disappears and the blue fluorescence is recovered. In a DMF solvent, fluorescent probe molecules are dispersed and far away from each other, effective intermolecular pi-pi accumulation effect cannot be formed between benzyl groups, the fluorescent probes do not aggregate, Aggregation Induced Emission (AIE) disappears, when Schiff base units and zinc ions are combined and coordinated in a 2:1 mode, a plurality of fluorescent probe molecules are close to each other, intermolecular pi-pi action is generated between the benzyl groups of the fluorescent probes, the fluorescent probes aggregate after coordination, and green AIE fluorescence is generated.

Schiff base structural units in the fluorescent probe HL molecule have the function of chelating metal ions, and can be coordinately combined with aluminum ions and zinc ions in the forms of 1:1 and 1: 2. The molecule has 3 benzyl structures, and fluorescent probe molecules can be aggregated through intermolecular pi-pi action after the fluorescent probe and zinc ions are coordinated in a 2:1 manner. The fluorescence probe generates fluorescence enhancement after the response of zinc and aluminum ions, the fluorescence enhancement and the concentration of the zinc and aluminum ions present a good linear relationship, and in addition, the Schiff base structure in the fluorescence probe HL is only in coordination combination with the zinc and aluminum ions and is not in coordination relationship with other metal ions.

The zinc-aluminum ion fluorescent probe HL has the advantages of double mechanisms, double responses, high sensitivity and specificity, no influence of other metal ions, easy cell uptake, low cytotoxicity and the like. The probe is applied to peripheral blood of a prostate cancer patient, can quantitatively detect the zinc content in serum, and provides valuable information for diagnosis of a suspected prostate cancer patient. The kit is combined with prostate cancer serum PSA detection, so that the diagnosis accuracy of prostate cancer can be effectively improved, the puncture diagnosis requirement of a patient with serum PSA in a gray scale interval is avoided, and the occurrence of over-diagnosis and over-treatment is further avoided.

Drawings

FIG. 1 is a schematic diagram of fluorescent probe synthesis.

FIG. 2 shows nuclear magnetic characterization of fluorescent probes.

FIG. 3a shows the change of ultraviolet spectrum after the response of fluorescent probe HL and zinc ions.

FIG. 3b shows the change of fluorescence spectrum after the response of fluorescent probe HL and zinc ions.

FIG. 3c Linear relationship between fluorescence intensity and zinc ion concentration after HL and zinc ion response.

FIG. 3d change of UV spectrum after HL and aluminum ion response.

FIG. 3e change in fluorescence spectra after HL and aluminium ion response.

FIG. 3f Linear relationship between fluorescence intensity and aluminum ion concentration after HL and aluminum ion response.

FIG. 4a HL and zinc ion recognition specificity study.

FIG. 4b effect of nonspecific ions on the fluorescence response between HL and zinc ions.

FIG. 4c JOB curves of HL and Zn ion coordination.

FIG. 4d HL and aluminum ion recognition specificity study.

FIG. 4e effect of nonspecific ions on the fluorescence response between HL and aluminum ions.

FIG. 4f JOB curves of HL and aluminum ion coordination.

FIG. 5 is a graph of the response of fluorescent probes and zinc aluminum ions in cells, where a: bright field, zinc response fluorescence and aluminum response fluorescence images after cells are directly incubated with a culture medium containing a fluorescent probe; b: and (3) bright field, zinc response fluorescence and aluminum response fluorescence images of cells which are incubated with a culture medium containing zinc and aluminum ions in advance and then incubated with a culture medium containing a fluorescent probe.

Figure 6 is the prostate cancer serum zinc test results for prostate cancer patients, normal humans, and prostate non-cancerous lesion patients. a is prostate cancer patient, b is normal person, c is prostate non-cancerous lesion, d is prostate cancer serum zinc test result of patient.

FIG. 7 shows the structural formula of a negative control molecule.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: synthesis and characterization of fluorescent probes

The synthesis of the ligand fluorescent probe HL is shown in figure 1. It is prepared from 3,4, 5-tribenzyloxy benzoyl hydrazine and salicylaldehyde through simple condensation reaction. The 3,4, 5-tribenzyloxy benzoyl hydrazine is prepared by two steps by using methyl gallate as an initial raw material, firstly, under the condition that potassium carbonate is used as an acid-binding agent, methyl gallate and benzyl chloride are subjected to substitution reaction to obtain 3,4, 5-tribenzyloxy methyl benzoate, then, 3,4, 5-tribenzyloxy methyl benzoate is subjected to hydrazinolysis to obtain the 3,4, 5-tribenzyloxy benzoyl hydrazine, and meanwhile, a ligand fluorescent probe is characterized by means of nuclear magnetism and the like to confirm that a very pure product is obtained, and the nuclear magnetism of the fluorescent probe is characterized as shown in figure 2.

Synthesis of ethyl 3,4, 5-tribenzyloxybenzoate (corresponding to a)

Ethyl 3,4, 5-trihydroxybenzoate (4.6g, 0.025mole) and potassium carbonate (12.42g, 0.09mole) were dissolved in 40ml anhydrous DMF and stirred at room temperature for 0.5 h. Benzyl chloride (10.4g, 0.081 moles) was then added to the reaction mixture. The solution is heated to 90-100 ℃ and stirred for 20 hours. After the reaction was completed, the reaction mixture was cooled to room temperature and poured into 250ml of pure water. The crude product was filtered and recrystallized from ethanol to give the final product as a white solid. Yield: 55%, melting point: 79 to 80 ℃.

Synthesis of 3,4, 5-Tribenzyloxybenzoyl hydrazine (corresponding to b)

Hydrazine hydrate (80%, 12mL) was added to a solution of ethyl 3,4, 5-tribenzyloxybenzoate (0.00445mol, 2g) in methanol (100 mL). The reaction mixture was stirred at 70 ℃ for 24 hours. White flocculent precipitate was observed, and the precipitate was filtered and washed with EtOH (30mL) 3-5 times to obtain the desired product. Melting point: 183 ℃ and 184 ℃.

Synthesis of 3,4, 5-Tribenzyloxy-benzoic acid (2-hydroxy-benzylidene) -hydrazide (i.e., fluorescent Probe HL, corresponding to c)

A solution of 3,4, 5-tribenzyloxybenzohydrazide (1mmol, 0.451g) in ethanol (30mL) was added to ethanol (20mL) containing salicylaldehyde (1mmol, 0.122 g). The mixed solution was then stirred at reflux for 12 hours and some white precipitate appeared. After cooling to room temperature, the mixture was filtered and recrystallized from ethanol and DMF to give the desired product. Yield: 83 percent. Melting point: 249 ℃ and 250 ℃.

Example 2: research on spectral behavior and specificity of fluorescent probe HL and zinc and aluminum ion response

A2 mM methanol solution of the fluorescent probe was prepared, and then diluted 100-fold to obtain a 20. mu.M methanol solution of HL. Various metal ion aqueous solutions with the concentration of 20 mu M are prepared by using different metal nitrates. Dropwise adding an aqueous solution of metal ions into the 20 mu M HL methanol solution, and recording the change of the ultraviolet absorption spectrum and the fluorescence emission spectrum of the methanol solution of the fluorescent probe. After the titration was completed, 20. mu.M of an aqueous solution of zinc and aluminum ions was continuously added dropwise to the mixed solution, and the change in fluorescence spectrum was further recorded. Meanwhile, adding a zinc and aluminum ion aqueous solution with continuous concentration into the HL solution with the concentration of 20 mu M, measuring the integral fluorescence intensity of the mixed solution under different zinc and aluminum ion concentrations, and constructing a zinc and aluminum ion concentration-fluorescence intensity standard curve. In addition, 1mL of 20-mu M fluorescent probe methanol solution is placed in a sample bottle, 20-mu M zinc and aluminum ion solution is slowly dripped, the fluorescence intensity of the system is continuously measured in the adding process, a JOB curve is drawn, and the ratio of the quantity of zinc and aluminum ions at the position with the strongest fluorescence intensity to the quantity of the fluorescent probe is determined, namely the coordination ratio of the fluorescent probe HL to the zinc-aluminum ions during coordination. The changes of the ultraviolet spectrum and the fluorescence spectrum after the fluorescent probe HL coordinates with zinc and aluminum ions and the linear standard curves of the fluorescence intensity and the ion concentration are shown in figures 3 a-3 f, and the results show that after the fluorescent probe HL responds to the zinc and aluminum ions, the ultraviolet absorption at the position of 300nmThe peak disappeared and a new absorption peak was generated at 400nm/385 nm. Taking the wavelength as the excitation wavelength of the probe after response to coordinate with the corresponding fluorescent probe-Zn²⁺The maximum fluorescence emission wavelength is 509nm, green fluorescence is generated by AIE effect, and a fluorescent probe HL-Al³⁺At 450nm, produces blue fluorescence by the PET effect. The coordination recognition specificity and JOB coordination curves of the fluorescent probe HL and zinc and aluminum ions are shown in figures 4 a-4 f, except for the zinc and aluminum ions, other non-specific ions basically do not coordinate with the fluorescent probe HL, the fluorescence of the fluorescent probe does not change obviously, the zinc and aluminum ions can respectively start the HL fluorescence through an AIE mechanism and a PET mechanism, and the fluorescence intensity and the ion concentration have good linear relation. The JOB curve shows that the coordination ratio of the fluorescent probe to zinc and aluminum ions is 2:1 and 1:1 respectively.

Example 3: zinc-aluminum ion response imaging and monitoring in cells

PC-3 prostate cancer group 2 was plated at a cell density of 8 ten thousand per well in 12-well cell culture plates and attached overnight. After the cells are attached to the wall, the old culture medium is removed, wherein 1 group is added with pure fresh culture medium for culture, and the other 1 group is respectively added with fresh culture medium of 20 mu M zinc ions and aluminum ions, and then the incubation is continued for 30 minutes. After the incubation was completed, the medium was removed, the cells were washed 2 times with fresh medium, and then the cells of group 2 were added again with 20 μ M of a fluorescent probe medium solution (DMSO being the most cosolvent, with a concentration of less than one thousandth), and the cells were incubated for another 30 minutes, after which the medium was removed, and after the cells were washed twice with PBS, the cells were observed using a fluorescence microscope, and the results are shown in fig. 5. Because the cells and the culture medium contain zinc, the cells generate obvious green fluorescence after being incubated with HL, and the fluorescence can be slightly increased by adding zinc ions. The aluminum ions serving as cytotoxic substances are extremely low in content in cells and cannot be detected, and after the aluminum ions are added, the cells show obvious blue fluorescence, so that the fluorescent probe HL can effectively and dynamically monitor the change of the aluminum content in the cells.

Example 4: prostate cancer peripheral blood serum zinc assay

Peripheral blood was collected in 20mL each of 13 prostate cancer patients, 10 non-cancerous diseased prostate cancer patients, and 15 normal patients. After high speed centrifugation, 3mL of each serum was obtained. Then adding 2mL of DMF into the serum, vortexing for 3min to precipitate biomacromolecules in the serum, centrifuging, mixing 200 mu L of supernatant with 20 mu M of fluorescent probe solution (dissolved in DMF: water in a volume ratio of 2:3), measuring the fluorescence intensity, determining the zinc content in the serum, and simultaneously determining the PSA content in the serum of patients with prostate cancer and non-cancerous lesions by using parallel samples. The results of the measurement of serum zinc content in each sample are shown in fig. 6, and the clinical information of the patients is shown in table 1. The experimental results show that. Based on the fact that pure serum PSA levels are difficult to test for accurate diagnosis of prostate cancer, there is still the possibility of non-cancerous lesions of the prostate within the PSA interval of 4-10 ng/mL or even > 10 ng/mL. At the same time, a single zinc level may not be completely indicative of prostate cancer, and there is a similar serum zinc level to normal serum in some prostate cancer patients (e.g., as shown in cases x and y). Therefore, the serum PSA content and the zinc content of the suspected prostate cancer patient are synchronously determined and used for the combined diagnosis of the prostate cancer, so that the diagnosis accuracy of the prostate cancer can be effectively improved, on one hand, the treatment time can be prevented from being delayed due to the occurrence of false positives, and on the other hand, the occurrence of false positives can be prevented so as to avoid over-diagnosis and over-treatment.

Table 1: clinical information of patients and healthy volunteers

Example 5: research on effect of tribenzyl structure in fluorescent probe HL molecular structure in AIE fluorescence generation process of zinc recognition

In order to determine the effects of three benzyl structures in HL in the processes of zinc ion recognition and AIE fluorescence generation, the benzyl structures are replaced by ethyl groups, and the other structures and the corresponding synthesis modes are unchanged, so that a negative control molecule is obtained, wherein the structure of the negative control molecule is shown in the following figure 7. The de-complex was mixed with zinc ions and was found to have no function of producing green AIE fluorescence in response to zinc ions. Therefore, it is reasonable to assume that the three benzyl structures in HL play an important role in identifying the process of generating AIE fluorescence by zinc ions. In the coordination process of zinc ions and HL, because two HL molecules coordinated with zinc are close to each other and have limited conformation, 3 benzyl groups in the molecules are positioned in the same plane, so that pi-pi accumulation effect is generated between the two HL molecules, and a minimum aggregation unit is formed. Then, the multiple aggregation units are further aggregated through intermolecular pi-pi accumulation again, and finally aggregation induced emission fluorescence (AIE) is generated.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.