A zinc ion fluorescent probe based on 8-aminoquinoline and its preparation method and application

Abstract

The present invention discloses a zinc ion fluorescent probe based on 8-aminoquinoline, its preparation method and application. The zinc ion fluorescent probe based on 8-aminoquinoline has the following structural formula. The present invention uses 8-aminoquinoline as a matrix, introduces an acetylamino group at position 2 and reacts with salicylaldehyde at position 8 to design and synthesize a highly selective zinc ion fluorescent probe. 2+ The fluorescent probe APYQ is not interfered by other metal ions, and its synthesis steps are simple and the purification process is easy. The fluorescent probe of the present invention has excellent detection limit (1.87nM) and response time (5s). These two indicators are better than other Zn 2+ The fluorescent probe of the present invention can be used to detect Zn with naked eyes in ethanol / HEPES buffer solution and on test paper. 2+ , can also be used to detect Zn in Hela cells 2+ Fluorescence imaging has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of small molecule fluorescent probes, and particularly relates to an 8-aminoquinoline-based zinc ion fluorescent probe and a preparation method and application thereof. Background Art

[0002] Zinc is the second most abundant transition metal element in the human body, and its optimal concentration in the human body is crucial for cell replication, nucleic acid metabolism, tissue repair, growth and development. A deficiency of zinc ions in the human body can lead to serious neurological diseases such as Alzheimer's disease. Similarly, an excess of zinc ions can cause Parkinson's disease. In addition, excessive zinc ions in the environment can lead to heavy metal ion pollution. The World Health Organization (WHO) stipulates that the concentration of zinc ions in drinking water should not exceed 76 μM. Therefore, it is of great significance to develop rapid and highly sensitive methods for zinc ion detection in the environmental and biological fields.

[0003] Currently, traditional zinc ion detection techniques primarily include atomic absorption spectroscopy (AAS), electrochemical analysis, and spectrophotometry. However, the application of these techniques is limited by factors such as high equipment costs, long detection times, low sensitivity, and complex procedures. Compared to these traditional techniques, fluorescence spectroscopy has attracted considerable attention due to its portability, ease of operation, rapid response, and real-time in situ detection.

[0004] In recent years, quinoline derivatives have been reported as fluorescent molecular probes, especially 8-aminoquinoline derivatives, which are often used to identify zinc ions. However, these reported zinc ion probes still have a lot of room for improvement and enhancement. On the one hand, the similar electronic configuration between cadmium ions and zinc ions poses a problem for the high selectivity recognition of zinc ions. On the other hand, the detection limit of these probes (10 -5 ~10 -7 mol·L -1 ) is high, which limits its application range. Therefore, how to develop a fluorescent probe with high selectivity, high sensitivity and rapid detection of zinc ions has become a challenging topic. Summary of the Invention

[0005] Purpose of the invention: In order to solve the problems existing in the prior art, the present invention aims to provide a zinc ion fluorescent probe based on 8-aminoquinoline and its preparation method and application. The fluorescent probe compound of the present invention is 2+ It has high selectivity, high sensitivity (1.87nM) and rapid detection (5s), and can identify and detect Zn with the naked eye. 2+ , can be applied to HeLa cell fluorescence imaging and has good application prospects.

[0006] Technical solution: In order to achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is as follows:

[0007] A zinc ion fluorescent probe based on 8-aminoquinoline, the structural formula of which is shown below:

[0008]

[0009] The present invention also provides a method for preparing the 8-aminoquinoline-based zinc ion fluorescent probe, comprising the following steps:

[0010]

[0011] (1) reacting 2-acetamido-8-nitroquinoline with a reducing agent to obtain the intermediate AAQ;

[0012] (2) The intermediate AAQ and salicylaldehyde are reacted under acidic conditions to obtain the zinc ion fluorescent probe APYQ.

[0013] As a specific embodiment, in step (1), the reducing agent is selected from iron; the solvent of the reaction is selected from acetic acid; the reaction is carried out under the protection of inert gas at 60-70°C for 1.5-2.5 hours; the molar ratio of the 2-acetamido-8-nitroquinoline to the reducing agent is 1:4-1:6.

[0014] As a specific embodiment, in step (1), the post-processing method of the reaction includes: filtration, washing, removing the solvent, extracting with ethyl acetate, drying, removing the solvent, and purifying the crude product by silica gel column, using ethyl acetate and petroleum ether = 1:2 to 1:3 (v / v) as eluent for separation to obtain the intermediate AAQ.

[0015] As a specific implementation scheme, in step (2), the acid in the acidic condition is selected from acetic acid; the solvent of the reaction is selected from anhydrous ethanol; the reaction is carried out under the protection of inert gas at 80-90° C. for 2.5-3.5 hours; the molar ratio of the intermediate AAQ to acetic acid is 1:1-1:3, and the molar ratio of the intermediate AAQ to salicylaldehyde is 1:1-1:1.2.

[0016] As a specific embodiment, in step (2), the post-treatment method of the reaction includes: filtering the precipitate, washing with anhydrous ethanol, and drying to obtain the zinc ion fluorescent probe APYQ.

[0017] As a more preferred embodiment, the method for preparing the 8-aminoquinoline-based zinc ion fluorescent probe comprises the following steps:

[0018] A method for preparing a zinc ion fluorescent probe based on 8-aminoquinoline is carried out according to the following steps:

[0019] (1) Preparation of AAQ: 2-acetamido-8-nitroquinoline was dissolved in acetic acid, iron powder was slowly added, and the mixture was stirred at 65°C for 2 h under nitrogen protection. The mixture was filtered and washed while hot, and the solvent was removed by distillation under reduced pressure. The mixture was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column and separated using ethyl acetate and petroleum ether in a ratio of 1:2 to 1:3 (v / v) as eluent to obtain the intermediate AAQ.

[0020] (2) Preparation of APYQ: Salicylaldehyde was dissolved in anhydrous ethanol, and then acetic acid was added as a catalyst. Under nitrogen protection, the mixture was refluxed at 85°C for 10 min. The intermediate AAQ was added and the mixture was refluxed at 85°C for 3 h. The precipitate was filtered, washed three times with anhydrous ethanol, and dried to obtain the fluorescent probe APYQ.

[0021] Its synthetic route is as follows:

[0022]

[0023] The present invention also provides application of the 8-aminoquinoline-based zinc ion fluorescent probe in zinc ion detection.

[0024] Finally, the present invention provides a method for detecting zinc ions, which comprises using the 8-aminoquinoline-based zinc ion fluorescent probe to detect zinc ions in a solution.

[0025] As a specific embodiment, the method includes detecting zinc ions in an ethanol / HEPES buffer solution; preferably, the pH of the ethanol / HEPES buffer solution is 7.4±0.5, wherein the volume ratio of ethanol to HEPES buffer solution is (7-9):(3-1).

[0026] As a specific implementation scheme, under the action of zinc ions, the solution containing the zinc ion fluorescent probe changes from colorless to yellow under visible light and from blue to green under ultraviolet light.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0028] (1) The present invention uses 2-acetamido-8-nitroquinoline as a raw material to synthesize a fluorescent probe, and the synthesis steps are simple and the product separation and purification process is easy.

[0029] (2) Fluorescent probe APYQ for Zn 2+ Highly selective and unaffected by Na + , K + , Li + , Ag + , Ba 2+ , Ca 2+ , Cd 2+ ,Co2+ , Fe 2+ , Cu 2+ , Mg 2+ , Ni 2+ , Pb 2+ , Hg 2+ , Mn 2+ , Al 3+ , Cr 3+ , Fe 3+ Interference from metal ions.

[0030] (3) In the range of 0-10 μM, Zn 2+ There is a good linear relationship between concentration and probe fluorescence intensity. The detection limit of the probe is as low as 1.87nM, and the response time is as low as 5s. Considering the detection limit and response time, the fluorescent probe APYQ has the best performance compared with the probes reported in the past five years.

[0031] (4) The fluorescent probe APYQ can identify and detect Zn with the naked eye 2+ , can be actually applied to ethanol / HEPES buffer solution and probe pre-treated test paper, showing obvious color change. In biological applications, adding Zn 2+ The color change of cell imaging before and after is obvious, which has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The intermediate AAQ of the present invention 1 H NMR (400 MHz, DMSO-d6) spectrum;

[0033] Figure 2 The intermediate AAQ of the present invention 13 C NMR (100 MHz, DMSO-d6) spectrum;

[0034] Figure 3 Mass spectrum of the intermediate AAQ of the present invention;

[0035] Figure 4 The fluorescent probe APYQ of the present invention 1 H NMR (400 MHz, DMSO-d6) spectrum;

[0036] Figure 5 The fluorescent probe APYQ of the present invention 13 C NMR (100 MHz, DMSO-d6) spectrum;

[0037] Figure 6 Mass spectrum of the fluorescent probe APYQ of the present invention;

[0038] Figure 7Ultraviolet spectra of the fluorescent probe APYQ of the present invention when different metal ions are added;

[0039] Figure 8 Fluorescence spectra of the fluorescent probe APYQ of the present invention when different metal ions are added;

[0040] Figure 9 The fluorescent probe APYQ of the present invention is added with different concentrations of Zn 2+ UV spectrum of

[0041] Figure 10 The fluorescent probe APYQ of the present invention is added with different concentrations of Zn 2+ Fluorescence spectrum of

[0042] Figure 11 The fluorescence intensity of the fluorescent probe APYQ at 535 nm is similar to that of Zn 2+ Linear relationship graph of concentration;

[0043] Figure 12 The fluorescent probe APYQ of the present invention detects Zn 2+ Response time graph of

[0044] Table 1 The fluorescent probe APYQ of the present invention and other Zn 2+ Probe structural formula, detection limit, and response time;

[0045] Figure 13 The fluorescent probe APYQ of the present invention and other Zn 2+ Probe detection of Zn 2+ Detection limit and response time performance comparison chart;

[0046] Figure 14 Color change diagram of the fluorescent probe APYQ of the present invention when different metal ions are added under natural light;

[0047] Figure 15 The fluorescent probe APYQ of the present invention was added with different concentrations of Zn under natural light. 2+ Color change diagram of

[0048] Figure 16 Color change diagram of the fluorescent probe APYQ of the present invention when different metal ions are added under ultraviolet light;

[0049] Figure 17 The fluorescent probe APYQ of the present invention was added with different concentrations of Zn under ultraviolet light. 2+ Color change diagram of

[0050] Figure 18 Selectivity and sensitivity color change diagram of the test paper pretreated with the fluorescent probe APYQ of the present invention under natural light;

[0051] Figure 19Selectivity and sensitivity color change diagram of test paper pretreated with the fluorescent probe APYQ of the present invention under ultraviolet light;

[0052] Figure 20 The fluorescent probe APYQ of the present invention detects Zn in Hela cells 2+ Cell imaging diagram. DETAILED DESCRIPTION

[0053] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with 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 making creative efforts shall fall within the scope of protection of the present invention.

[0054] Example 1

[0055] A method for preparing a zinc ion fluorescent probe based on 8-aminoquinoline comprises the following steps:

[0056] (1) 0.4 g of 2-acetamido-8-nitroquinoline was added to a 100 mL round-bottom flask and dissolved in 50 mL of acetic acid. 0.72 g of iron powder was slowly added. Under nitrogen protection, the round-bottom flask was placed in an oil bath and stirred at 65° C. for 2 h. After hot filtration and washing, the solvent was removed by distillation under reduced pressure. The mixture was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column using ethyl acetate and petroleum ether = 1:3 (v / v) as eluent for separation to obtain 0.31 g of white powder intermediate AAQ with a yield of 89.1%;

[0057] (2) 0.15 g of salicylaldehyde was added to a 50 mL round-bottom flask and dissolved in 15 mL of anhydrous ethanol. Then, 150 μL of acetic acid was added as a catalyst. Under nitrogen protection, the round-bottom flask was placed in an oil bath and refluxed at 85°C for 10 min. 0.2 g of the intermediate AAQ was added and refluxed at 85°C for 3 h. The precipitate was filtered, washed three times with anhydrous ethanol, and dried to obtain 0.22 g of orange-red powder fluorescent probe APYQ with a yield of 73.8%.

[0058] Example 2

[0059] The fluorescence probe APYQ obtained in Example 1 is sensitive to Zn 2+ The selectivity includes the following steps:

[0060] Prepare a 1mM fluorescent probe APYQ stock solution with anhydrous ethanol, and prepare 10mM aqueous solutions of various metal ions with distilled water. Take 30μL of the fluorescent probe stock solution into a cuvette each time, add 30μL of various metal ion aqueous solutions in sequence, and then dilute the solution to 3mL with ethanol / HEPES (v / v=8 / 2, HEPES=10mM, pH=7.4) buffer solution. Mix well and use for UV and fluorescence testing, such as Figure 7 and Figure 8 As shown. Figure 7 Only when Zn is added 2+ and Cd 2+ The UV spectrum changes, and Zn 2+ The changes are more obvious; Figure 8 Only when Zn is added 2+ The fluorescence spectrum showed a significant enhancement, while the addition of other ions did not cause significant fluorescence changes. 2+ Shows excellent selective recognition ability.

[0061] Example 3

[0062] The fluorescence probe APYQ obtained in Example 1 is sensitive to Zn 2+ The concentration titration experiment includes the following steps:

[0063] Each time, 30 μL of 1 mM fluorescent probe stock solution was taken into the cuvette, and different concentrations of Zn 2+ Then dilute the solution to 3 mL with ethanol / HEPES (v / v=8 / 2, HEPES=10 mM, pH=7.4) buffer solution, mix well, and use it for UV and fluorescence testing. Figure 9 and Figure 10 As shown. 2+ With the increase of concentration, the UV peak at 434nm and the fluorescence peak at 535nm gradually increased; 2+ There is a good linear relationship between the concentration and the fluorescence emission peak intensity value, such as Figure 11 As shown. The linear equation is: y = 80.0671x + 79.3784, the linear correlation coefficient R 2 =0.99695, the probe pair Zn 2+ The detection limit of the probe was 1.87 nM, indicating that the probe was sensitive to Zn 2+ Has high sensitivity.

[0064] Example 4

[0065] The fluorescence probe APYQ obtained in Example 1 is sensitive to Zn 2+ The response time includes the following steps:

[0066] Take 30 μL of 1 mM fluorescent probe stock solution in a cuvette, add 30 μL of 10 mM Zn 2+ Then, dilute the solution to 3 mL with ethanol / HEPES (v / v=8 / 2, HEPES=10 mM, pH=7.4) buffer solution for fluorescence testing. Figure 12 As shown. Add Zn 2+ After that, the fluorescence intensity reached the highest within 5s, indicating that the probe can quickly respond to Zn 2+ .

[0067] Example 5

[0068] The fluorescent probe APYQ obtained in Example 1 and other Zn 2+ Probe detection of Zn 2+ The detection limit and response time performance comparison includes the following steps:

[0069] The Zn detection data in the past five years are listed. 2+ The probes with excellent performance are shown in Table 1. The detection limit and response time of probe APYQ are compared with them. Figure 13 As shown, it can be seen that the probe APYQ has the best comprehensive performance in terms of high sensitivity and fast response capability.

[0070] Table 1 Fluorescent probe APYQ and other Zn 2+ Comparison of probes

[0071]

[0072]

[0073]

[0074] Example 6

[0075] The fluorescent probe APYQ obtained in Example 1 can recognize Zn with naked eyes in solution. 2+ The experiment includes the following steps:

[0076] Each time, 10 μM fluorescent probe mother solution was taken into the culture bottle, and 100 μM aqueous solutions of various metal ions were added in sequence. Then, ethanol / HEPES (v / v=8 / 2, HEPES=10mM, pH=7.4) buffer solution was taken to dilute the solution to 3 mL and mixed evenly. Figure 14 and Figure 15 As shown. Figure 14 In visible light, only Zn 2+ The color of the solution changes from colorless to bright yellow; Figure 15 In the ultraviolet light, Zn 2+ The solution color changed from blue to bright green. Further tests were performed by adding different concentrations of Zn2+ ,like Figure 16 and Figure 17 As shown, the color changes gradually under visible light and ultraviolet light, indicating that Zn can be identified with the naked eye. 2+ .

[0077] Example 7

[0078] The fluorescent probe APYQ obtained in Example 1 can identify Zn with naked eyes in the test paper. 2+ The experiment includes the following steps:

[0079] Prepare a 1mM fluorescent probe APYQ stock solution with anhydrous ethanol, soak the test paper in the probe stock solution for one day, and then dry the test paper for subsequent testing. Add 1mM concentration of various metal ion aqueous solutions on the test paper and add different concentrations of Zn 2+ On the test paper, dry it and take a comparison photo, such as Figure 18 and Figure 19 As shown. Figure 18 Under visible light, only when Zn is added 2+ The color changes from colorless to yellow, and the color changes gradually under the corresponding sensitivity; Figure 19 Under medium ultraviolet light, only Zn 2+ The color changes from blue to green, corresponding to the gradual change in color under sensitivity, indicating that the probe can be used for "naked eye" identification and quantitative detection of Zn 2+ .

[0080] Example 8

[0081] The fluorescent probe APYQ obtained in Example 1 detects Zn in cells 2+ The fluorescence imaging experiment includes the following steps:

[0082] HeLa cells were seeded onto a confocal dish and incubated in a 37°C incubator containing 5% carbon dioxide for 24 hours to allow the cells to adhere. The adherent cells were stained with the probe APYQ (10 μM) for 30 minutes, washed three times with phosphate buffered saline (PBS, pH = 7.4) to remove excess dye, and then photographed using a confocal microscope. Zn was then added. 2+ (10 μM) and incubated for 30 min, and photographs were taken, e.g. Figure 20 As shown. It can be seen that the addition of Zn 2+ After that, there is obvious green fluorescence, which shows that the probe APYQ has good cell permeability and has good application prospects in the biological field.

[0083] The above describes the implementation of the present invention in detail with reference to specific embodiments. However, the present invention is not limited to the above implementation. Various changes can be made within the scope of knowledge possessed by ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A zinc ion fluorescent probe based on 8-aminoquinoline, the structural formula of which is shown below:

2. The method for preparing the 8-aminoquinoline-based zinc ion fluorescent probe according to claim 1, wherein The following steps are involved: (1) reacting 2-acetamido-8-nitroquinoline with a reducing agent to obtain the intermediate AAQ; (2) The intermediate AAQ and salicylaldehyde are reacted under acidic conditions to obtain the zinc ion fluorescent probe APYQ.

3. The method for preparing a zinc ion fluorescent probe based on 8-aminoquinoline according to claim 2, wherein In step (1), the reducing agent is selected from iron; the solvent of the reaction is selected from acetic acid; the reaction is carried out under the protection of an inert gas at 60 to 70° C. for 1.5 to 2.5 hours; and the molar ratio of the 2-acetamido-8-nitroquinoline to the reducing agent is 1:4 to 1:

6.

4. The method for preparing a zinc ion fluorescent probe based on 8-aminoquinoline according to claim 2, wherein In step (1), the post-processing method of the reaction includes: filtering, washing, removing the solvent, extracting with ethyl acetate, drying, removing the solvent, and purifying the crude product by silica gel column, using ethyl acetate and petroleum ether with a volume ratio of 1:2 to 1:3 as eluent for separation to obtain the intermediate AAQ.

5. The method for preparing a zinc ion fluorescent probe based on 8-aminoquinoline according to claim 2, wherein In step (2), the acid in the acidic condition is selected from acetic acid; the solvent of the reaction is selected from anhydrous ethanol; the reaction is carried out under the protection of inert gas at 80-90° C. for 2.5-3.5 hours; the molar ratio of the intermediate AAQ to acetic acid is 1:1-1:3, and the molar ratio of the intermediate AAQ to salicylaldehyde is 1:1-1:1.

2.

6. The method for preparing a zinc ion fluorescent probe based on 8-aminoquinoline according to claim 2, wherein In step (2), the post-processing method of the reaction includes: filtering the precipitate, washing with anhydrous ethanol, and drying to obtain the zinc ion fluorescent probe APYQ.

7. The use of the 8-aminoquinoline-based zinc ion fluorescent probe according to claim 1 in zinc ion detection, characterized in that: The application does not involve using a living human / animal body as a direct implementation object for diagnosing the cause of disease / treating disease.

8. A method for detecting zinc ions, characterized in that, The invention comprises using the 8-aminoquinoline-based zinc ion fluorescent probe according to claim 1 to detect zinc ions in a solution, wherein the detection method does not involve a living human body or animal body as a direct implementation object and is used for diagnosing the cause of disease or treating the disease.

9. The detection method of zinc ion according to claim 8, wherein The method involves detecting zinc ions in an ethanol / HEPES buffer solution.

10. The method for detecting zinc ions according to claim 9, wherein The pH of the ethanol / HEPES buffer solution is 7.4±0.5, wherein the volume ratio of ethanol to HEPES buffer solution is (7-9):(3-1).

11. The method for detecting zinc ions according to claim 8, wherein Under the action of zinc ions, the solution containing the zinc ion fluorescent probe changes from colorless to yellow under visible light and from blue to green under ultraviolet light.

Images